STUDY ON CHEMICAL AND BIOLOGICAL ACTIVITIES OF HYDNOPHYTUM FORMICARUM EXTRACTS AND THEIR APPLICATIONS IN BISCUITS
10 Truong Thi Minh Hanh, Nguyen Thi Bich Thao STUDY ON CHEMICAL AND BIOLOGICAL ACTIVITIES OF HYDNOPHYTUM FORMICARUM EXTRACTS AND THEIR APPLICATIONS IN BISCUITS Truong Thi Minh Hanh1, Nguyen Thi Bich Thao2 University of Science and Technology, The University of Danang; tminhhanh2001@yahoo.com; Hue industrial College; nguyenbichthao04h2b@gmail.com Abstract - Hydnophytum formicarum is a medicinal plant that has a variety of bioactive compounds and has been traditionally used for therapeutic purposes Hydnophytum formicarum grows on the stems of large trees and in a symbiosis with a type of ants This study investigates the effects of technological factors on the extraction process of Hydnophytum formicarum in n-hexan, ethyl acetate, and ethanol solvents by using the Soxhlet method Our study also identified the best extraction conditions for each solvent Several major constituents present in the extracts were identified by using the GC-MS In addition, investigation results about the biological activity showed that all of the three extracts had high antioxidant and antimicrobial activity Therefore, in order to produce new biscuits that would be healthy for adults and have long preservation duration, we added Hydnophytum formicarum ethanol extract The amount of Hydnophytum formicarum ethanol extract used in biscuit formula was gram of condensed extract/100 grams of dough In sensory evaluation of the produced biscuits using consumer acceptance tests, 50 students in Danang University evaluated their preferences for color, flavour, taste, and general acceptance The obtained results have shown that statistically consumers will be likely to accept the produced biscuits Key words - sensory evaluation; antioxidant activity; antimicrobial activity; Hydnophytum formicarum; acceptance test Introduction Kien ki nam has scientific name of Hydnophytum formicarum Andis, it is also called “cay o kien” or “bi ky nam” This plant has been recently discovered by residents of Highland Central region and is used to treat several diseases related to liver, kidney, yellow skin, cough, and joint inflammation [1] In Asian countries such as Indonesia, Philippine, Thailand, and Malaysia, Hydnophytum formicarum is also used as traditional medicine to cure diseases such as headache, stomach pain, joint pain, liver inflammation, and cancers [2] Hydnophytum formicarum has valuable ingredients and compositions such as flavonoid, vitamin E, and mineral salts, which heighten the value of this medicinal plant to human health [3] However, research on chemical components and biological activities of Hydnophytum formicarum in Vietnam has received little attention Therefore, research on chemical components and biological activities of Hydnophytum formicarum in extracted solutions has important scientific contributions and practical applications Research results would lay scientific foundations for evaluating medicinal plant resources in Vietnam and preserving biological diversity as well as exploiting and utilizing this precious gene to improve community health The aims of “study on chemical and biological activities of Hydnophytum formicarum extracts and their supplemental applications in biscuits” were as follows: - Study on element that affect Hydnophytum formicarum extraction process in n-hexane, ethyl acetate, and ethanol solutions; - Identification of several chemical ingredients of Hydnophytum formicarum; - Identification of biological activities of Hydnophytumformicarum; - Supplement Hydnophytum formicarum to biscuits to create new products that benefit adults’ health; - Sensory evaluation of the produced biscuits using consumer acceptance tests Raw material and research methods 2.1 Raw material Hydnophytum formicarum roots were collected at Kbang District, Gia Lai Province The roots were then washed, dried out, ground into powder and stored at room temperature 2.2 Research methods - Extraction method using soxhlet system The necessary amount of Hydnophytum formicarum powder was weighed, wrapped in filter paper, and put in the body of soxhlet system 170 ml of n-hexane or ethyl acetate or ethanol solvent was added to a 500 ml round bottom flask that has already been washed and dried out before The flask was then attached to the body of soxhlet system A pipe cooler system was also attached to the body of the soxhlet system The compound were then extracted at an appropriate temperature for each solvent (70oC for n-hexane, 80oC for ethyl acetateand 80oC for ethanol) Subsequently, the obtained solvent was filtered to obtain extracted solvent and residual The extracted solvent was then dried out to reduce liquid solution The obtained solvent was poured into high temperature tolerated glass cups, which were already dried out and marked with ordered numbers These glass cups were placed in a dryer at an appropriate temperature for each solvent until dry extract was acquired After that, the cups were removed from the dryer When the cups cooled down, they were placed into an exsiccator until they reach the room temperature The cups were then measured using analytical measure and their weights were recorded The chemical ingredients of the extract identified at the best condition was then investigated by using GC-MS method at Quatest 2-02 Ngo Quyen Street, Son Tra District, Da Nang City The distillation process to obtain the desired solven was performed on distillation flask in order to reuse the solvent for the next extraction process The formula for computing extraction yield is as THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(91).2015 follows: 𝑂𝑏𝑡𝑎𝑖𝑛𝑒𝑑 𝑑𝑟𝑦 extract𝑚𝑎𝑠𝑠 Extraction yield= 𝑅𝑎𝑤𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙𝑚𝑎𝑠𝑠 x 100 (%) - The antibacterial activity of the plant extracts was determined using the disc-diffusion method [4]; - The diameter of the clear zone: ̅̅̅̅ ∑𝑛 𝑖=1(∆𝐷𝑖−∆𝐷 ) ΔD = D – d (mm) s = √ With 𝑛−1 D: the diameter of clear zone of inhibition (mm); d: the diameter of hole (mm); ΔDi: the diameter of clear zone in experiment I; ̅̅̅̅ ∆𝐷 : the average diameter of clear zones; 11 extracted in solvent for 2h, 4h, 6h, and 8h respectively with the extracting temperature of 700C According to the Figure 1, the extraction yield was higher when the extracting time increased from to hours but remained unchanged when reached hours In the initial stage, the solubility of material was still high so the yield also increased However, there were no more soluble compounds to be extracted with longer extracting time In addition, some extracted soluble compounds may be volatile subtances so the amount of extracted compounds would decreased in longer extracting time There was no significant difference of the yield in the and 6-hour experiments, therefore the extracting time would be hours in next experiments s: sample standard deviation; n: the number of experiments - Statistical analysis: The mean and the standard deviation were calculated based on duplicate experiments The Minitab was used for statistical analysis Results and Discussion 3.1 Studying the effects of extracting time on the Hydnophytum formicarum extraction in different extracting solvents According to Hanh et al [9], the Hydnophytum formicarum extracts contained high amount of flavonoids (25.37%) compared to the extracts from centalla (4.5%), tea (4.41%) [6], heartleaf (2.73%) [7], having proven effective in treating some diseases This plant also contained protein, fat, starch, sugar with low amount that used for the diet The high percentage of cellulose had also contributed to improve the treatment of cancer, obesity, diabetes, etc The metals from Hydnophytum formicarum were proven to add more minerals to support the biometabolism in body Free radical scavenging was evaluated using the DPPH free radical that indicated the Hydnophytum formicarum extracts had the antioxidant activity of 10.69% compared to resveratrol In this project, the extraction conditions of the Hydnophytum formicarum in some extracting solvents as n-hexane, ethyl acetate, ethanol and their chemical constituents were studied 3.1.1 Studying the effects of extracting time on extraction yield of n-hexane extract The grams samples of powdered materials were Extraction yield (%) - Sensory Evaluation Methods: In consumer acceptance tests, 50 students (33 females, 27 male) at Danang University who are regular biscuit consumers joined our study The location of the tests was the Sensory Evaluation Laboratory at Danang University; 1.20 1.125 1.138 1.138 1.15 1.051 1.10 1.05 1.00 0.95 Extraction time (h) Figure The effects of extraction time on extraction yield (n-hexane extracts) To conclude, the optimum extraction conditions were found to be grams of material in extracting time of hours for n-hexane solvent, extraction yield is 1.125% 3.1.2 Studying the effects of extracting time on extraction yield of etyl acetate and ethanol extracts Extraction yield (%) - The antioxidative activity assay: The DPPH free radical scavenging method [5]; - The Gas chromatography-mass spectrometry (GCMS): To identify chemical compounds in different extracts; - Determination of flavonoid contents by using the B.C Tali method [6, 7]; 6.433 6.525 4.046 2.002 2 Extraction time (h) Figure The effects of extraction time on extraction yield (etyl acetate extracts) The experiments with ethyl acetate and ethanol extracts were conducted in the same conditions of n-hexane extract with the change of extracting temperature to 800C The results were shown in the 12 Truong Thi Minh Hanh, Nguyen Thi Bich Thao Extraction yield (%) Figures and 3.The optimum extraction conditions were found to be grams of material in extracting time of hours for these both solvents Among the solvents ethanol achieved the highest extraction yield (13.753%), n-hexan achieved the lowest extraction yield (1.125%) 13.753 13.853 14 13 12 12.460 11.732 11 10 Extraction time (h) Figure The effects of extraction time on extraction yield (ethanol extracts) 3.2 Identification the chemical constituents of the extracts in different solvents The gas chromatography-mass spectrometry (GC-MS) was used to identify chemical compounds in different extracts The results from the chromatogram were compared to the standard library determining the wide range of chemical constituents of these extracts The major chemical constituents were shown in the Table 1, and The Table shows that the n-hexane extract had the highest amount of 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester(79.55%) following by vitamin E (1.03%) The other constituents made up under 1% The 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester (79.55%) have proven effective in antioxidant and anti-inflammatory activity [10] The fatsoluble vitamin E is an antioxidant that help to reduce the aging and enhance the metabolism The Table shows that the etyl acetate extract had the highest amount of 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester (57.81%) following byresorcinol (17.22%) The quantity of vitamin E in this extract was higher than in n-hexane one (2.29%) and the other constituents made up under 1% The resorcinol is known as an antibacterial, antifungal compound and used to treat seborrheic dermatitis, acne, eczema, psoriasis and other skin disorders [11], [12] The Table shows that the ethanol extract had the highest amount of resorcinol (48.12%) following by 1,2Benzenedicarboxylic acid, mono (2-ethylhexyl) ester (0.89%) Some compounds could not be identified and named Table The chemical constituents were contained in n-hexan extracts No Retention time (mins) Name- Molecular formula Chemical structure 21.419 HeptadecaneC17H36 0.08 23.926 OctadecaneC18H38 0.19 26.365 NonadecaneC19H40 0.31 28.723 EicosaneC20H42 0.42 31.055 33.990 37.787 Tetracosane C24H50 0.49 38.995 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester C16H22O4 79.55 42.336 Vitamin E 1.03 Heneicosane C21H44 Docosane C22H46 (%) 0.46 0.45 Table The chemical constituents were contained in etyl acetate extracts No Retention time (mins) Name- Molecular formula 14.392 Resorcinol Chemical structure (%) 17.22 THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(91).2015 13 26.364 Naphthalene, 1,3-dimethyl 0.11 28.718 NonadecaneC19H40 0.17 31.057 HeneicosaneC21H44 0.23 37.783 EicosaneC20H42 0.23 38.971 42.329 42.336 Hexanedioic acid, bis (2-ethylhexyl0 ester 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester C16H22O4 1.08 57.81 Vitamin E 2.29 Table The chemical constituents were contained in ethanol extracts No Retention time (mins) 11.530 38.955 Name- Molecular formula Chemical structure (%) Resorcinol 48.12 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester C16H22O4 3.3 Studying the biological activities of the extracts 3.3.1 The antibacterial activites The extracts of Hydnophytum formicarum exhibited antibacterial activity against Bacillus subtilis and Staphylococcus aureus and the 10-4 dillution were chosen to observe the results The results were shown in Table indicating that all three plant extracts have antibacterial effects The diameter of clear zone of inhibition from etyl acetate extract was biggest, it means that this extract had highest antibacterial activities Table The diameter of clear zone of inhibition against Bacillus subtilis and Staphylococcus Sample Diameter (mm) (against Bacillus subtilis) Diameter (mm) (against Staphylococcus) Hexane extract 3.07±0.06 2±0.27 Etyl acetate extract 15±0.45 12±0.58 Ethanol extract 13±0.31 8±0.33 Control sample (distilled water) 0 3.3.2 The antioxidant activites The three extracts of Hydnophytum formicarum were concentrated to plant jelly and analyzed at Bioactive Compounds Laboratory-Institute of natural products chemistry, Hanoi The results are shown in Table 0.89 Table The results of antioxidant activities from three plant extracts No Sample The substrate The ratio of concentration to the extracts produce 50% to the reduction of the standard DPPH EC50 (µg/ml) Ethanol extract 17.4 2.1 Etyl acetate extract 13.5 1.62 Hexane extract >128 >15.4 Resveratrol 8.3 The resveratrol, the antioxidant with highest activity, was found in red grapes The resveratrol antioxidant activity is higher seven times than vitamin E and ten times than vitamin C [13] According to Table 5, the resveratrol concentration to produce 50% reduction of the DPPH was 8.3 µg/ml The ethanol extract concentrate to produce 50% reduction of the DPPH was17.4 (µg/ml) that is lower than 2.1 times than the resveratrol antioxidant whereas this number in ethyl acetate was 13.5 ml being lower than the resveratrol 1.26 times and much higher than the water extracts [9] It is determined that the antioxidant activity of both extracts was quite high and the ethyl acetate had the strongest activity The n-hexane extract only showed its weak antioxidant activity at the concentration higher than 128 (µg/ml) 14 Truong Thi Minh Hanh, Nguyen Thi Bich Thao 3.4 Application of the Hydnophytum formicarum extracts for production of biscuits According to the above results, the ethyl acetate and ethanol extracts contained chemical compounds and had high antioxidant activities, therefore the ethanol extract was chosen to be concentrated and applied for production of biscuits The biscuits added Hydnophytum formicarum extract is good for health and have specific brown color These biscuits also could be preserved in longer time because of the high antibacterial and antioxidant activities 3.4.1 The ratio of ingredients The ingredients include flour, sugar, corn starch, eggs, leavens, butter The production of biscuits in laboratory scale as following steps: preparing ingredients, preparing the emulsion, mixing, shaping, cooling, packaging, sterilization with UV, making final products, storing The Hydnophytum formicarum added ratio was gram/100 grams dough based on the results from ranking test of sensory evaluation 3.4.2 The quality characteristics of final product The results of chemical composition and biological test were shown in Table and Table The chemical composition of biscuits being added Hydnophytum formicarum No Chemical composition Unit Amount Water content % sample weight 4.0 Flavonoid % sample weight 0.595 Fats % sample weight 13.4 scores below as null hypotheses The results of the consumer acceptance test were analyzed by the Minitab 16.2 and shown in Table Table One-sample z-tests of consumer acceptance tests for biscuits product Color Smell Taste General acceptance Null mean 5 5 Mean 5,900 ± 1.502 6.900 ±1.035 6.400±1.178 6.360 ± 1.083 p value 0.000 0.000 0.000 0.000 Score: 1: extremely dislike, 2: very dislike, 3: dislike, 4: slightly dislike, 5: neutral, 6: slightly satisfied, 7: satisfied, 8: very satisfied, 9: extremely satisfied The study was conducted with the confidence intervals of 95% According the p value, the probabilities of obtaining the mean values lower than for the mentioned criteria are zero Therefore, we accept that the population mean values of these acceptance norms are always higher than In addition, the standard deviation of color norm was 1.502 that was much higher than other sensory norms, it means that the acceptance of consumers to product’s color had the high variation among consumers The smell norm was highly accepted and had low variation as the low standard deviation (1.035) The taste and general acceptance norms indicated the normal satisfaction of consumers Table The results of biological test of biscuits No Name Unit Reference Result TCVN 4882:2007 Not observed Total Coliform count MPN/g Escherichia coli MPN/g TCVN 6846:2007 Total yeast count CFU/g ISO 21527:2008 Total mold count CFU/g ISO 21527:1008 Not observed