Optimization of extraction of bioactives from sea buckthorn (Hippophae rhamnoides L.) woody waste

` Statement of the supervisor I, Dr. Erika Vági supervisor, hereby declare that the thesis written by Sergio Alonso Muñoz, (Neptune code: BEWOK8), titled: Optimization of extraction of bioactives from sea buckthorn (Hippophae rhamnoides L.) woody waste is his own writing prepared under my supervision. I also declare that the thesis meets the formal and professional requirements of the Budapest University of Technology and Economics and those of the Faculty of Chemical Technology and Biotechnology, thus I support its submission. 06/6/2019 Budapest (supervisor) Statement of the student I, Sergio Alonso Muñoz (Neptun code: BEWOK8) as author of the thesis hereby declare that my thesis titled: Optimization of extraction of bioactives from sea buckthorn (Hippophae rhamnoides L.) woody waste, is my original writing and I have not plagiarised any other work. All third party materials including published and unpublished sources were referenced. I acknowledge that the intellectual property rights of the methods used and the results of any research or development described in the thesis belong to the participating researchers and institutions/companies, thus their utilization or publication must not be initiated before the approval of all parties. I also declare that during the preparation and writing the thesis I did not mislead my supervisor(s) and thesis advisor. 06/6/2019 Budapest. (student) 2 Acknowledgement I want to thank my parents first of all for all the unconditional support they have given me, without them none of this would have been possible. I want to thank the universities from both Budapest and Valladolid for their great work. And especially I want to thank Dr. Erika Vági, thank you for all the effort and dedication you have put in me. 3 BSc. Thesis Optimization of extraction of bioactives from sea buckthorn (Hippophae rhamnoides L.) woody waste Written by: Sergio Alonso Muñoz BSc. in Chemical Engineering Supervisor: Dr. Erika Vági assistant professor 2019. 4 Table of contents 1. Introduction...................................................................................................................... 6 2. Objectives ......................................................................................................................... 7 3. Theoretical background .................................................................................................... 8 3.1 Sea buckthorn (Hippophae rhamnoides L.) ..................................................................... 8 3.1.1. Uses and benefits of sea buckthorn ......................................................................... 9 3.1.2. Cosmetical usage ..................................................................................................... 9 3.1.3. Health benefits ..................................................................................................... 11 3.2. Extraction methods ...................................................................................................... 12 3.2.1. Stirred tank extraction ........................................................................................... 12 3.2.2 Soxhlet extraction ................................................................................................... 13 3.2.3. Extraction solvents ................................................................................................ 15 3.3. Antioxidants .................................................................................................................. 15 3.3.1.Determination of antioxidant activity by means of DPPH (2,2-diphenyl-1picrylhydrazyl) ................................................................................................................. 17 3.3.2. Study of the oxidative capacity of oils by Rancimat method ................................... 18 4. Materials and methods ...................................................................................................... 20 4.1. Material and chemicals .................................................................................................. 20 4.2. Methods ....................................................................................................................... 21 4.2.1. Determination of moisture content ....................................................................... 21 4.2.2. Particle size distribution......................................................................................... 22 4.2.3. Stirred tank extraction ........................................................................................... 23 4.2.4. Soxhlet extraction .................................................................................................. 29 4.2.5. Antioxidant activity of samples .............................................................................. 30 4.2.5.1 Antioxidant activity measured by DPPH method .................................................. 30 4.2.5.2. Study of protective effect of the extract by Rancimat method ............................ 33 5. Results & discussion ........................................................................................................... 36 5.1. Characterization of sea buckthorn stalks......................................................................... 36 5.2. Results of extraction ...................................................................................................... 37 5.2.1. Effect of temperature on the extraction yield ........................................................ 40 5.2.2. Effect of water content of EtOH-water extraction solution ..................................... 42 5.2.3. Repeatability of experiments ................................................................................. 44 5.2.4. Re-extraction of extracted residue ......................................................................... 44 5.3 Antioxidant activity of sea buckthorn extracts .................................................................. 44 5.3.1 Antioxidant activity of sea buckthorn extracts by DPPH method ............................. 44 5 5.3.2. Protective effect of sea buckthorn extracts evaluated by Rancimat method .......... 47 6. Conclusion .......................................................................................................................... 50 7. References ...................................................................................................................... 52 6 1. Introduction Nowadays products from sea buckthorn can be easily found as the berry of this easily grown, widely distributed shrub is very important source of vitamins, minerals, natural antioxidants, amino acids, fatty acids and other bioactive substances. As a natural medicine it has potential for arthritis, gastrointestinal ulcers, gout and skin rashes and irritations [1-4]. The fruits of sea buckthorn can also be used in the day to day to make jams, juices or oils. The fruit, leaves and seeds of sea buckthorn has been used for treatment of skin disorders, stomach malfunctioning, thrombosis, hepatic injuries, in Asian traditional medicine. The leaves of sea buckthorn are rich in phenolic compounds and possess strong antioxidant activity [3-6]. The stalk or stem of sea buckthorn is the part of the plant that is going to be investigated in this project. The stalks until now have been considered as a waste. The main objective of the research is studying the antioxidant activity of the extracts of stem and branches of sea buckthorn, using different extraction methods such as stirred tank extraction and Soxhlet extraction in laboratory scales with different solvents such as pure ethanol, water, ethanol-water solutions and n-pentane. The optimization of extraction conditions, such as water content in the ethanol-water solutions, the extraction temperature is the main focus of this work for producing an extract which has strong antioxidant activity. The antioxidant activity of extracts is analysed by the DPPH method using spectrophotometer, in which the hydrogen-donor activity of extracts can be revealed in the presence of 2,2-diphenyl1-picrylhydrazyl (DPPH) free radical. On the other hand, the protecting effect of the extracts are also revealed by Rancimat fast oxidative measurement. Mixing together the extract with sunflower seed oil the extract might act as a protective agent which protect the oil from oxidation, therefore the shelf-life of oil can be increased. The analysis is carried out using Rancimat appartus. As a result of this work an optimized extraction method will be pointed out providing extract in high yield and with high antioxidant activity. 7 2. Objectives 1. Determination of the moisture content of samples. 2. Determination of the particle size distribution of the plant material. 3. Extraction of stalks using two different methods: 3.1 Stirred tank extraction with different mixtures of ethanol and water and at different temperatures:  96% Ethanol at 40⁰C, and 60⁰C  70% Absolute ethanol 30% Distilled water (V/V) at 40⁰C, 60⁰C, 80⁰C  50% Absolute ethanol 50% Distilled water (V/V) at 40⁰C, 60⁰C, 80⁰C  30% Absolute ethanol 70% Distilled water (V/V) at 40⁰C, 60⁰C, 80⁰C  100% Distilled water at 40⁰C, 60⁰C, 80⁰C temperatures. 3.2 Soxhlet extraction with two different solvents:  96% Ethanol  n-pentane 4. Determination of the antioxidant activity of samples with the DPPH method. 5. Analyse the oxidative capacity of the samples. 6. Compare the results and drawn conclusion. 8 3. Theoretical background 3.1 Sea buckthorn (Hippophae rhamnoides L.) Sea buckthorn, also known Hippophae rhamnoides L., is a deciduous shrub within the genus Hippophae, belonging to the family Elaeagnaceae. It is native to a large area of Europe, Asia, Mongolia and Canada [1]. They reach heights of 0.5 to 6 m; rarely as high as 18 m in Central Asia and are generally found in dry, sandy areas. They are tolerant to salt in the air and soil but have full sun exposure requirements for full development and do not tolerate shade conditions near large trees. The name of the plant comes precisely from the fact that they grow close to the sea and withstand extreme conditions from -40⁰C to 40⁰C degrees [2]. Sea buckthorn has fruits those are rich in essential nutrients such as fatty acids, vitamins -A, -C, -E and a wide variety of bioactive compounds such as antioxidants and phenolic compounds. The berries are typically used to make oils and to treat diseases of the digestive tract [3]. The leaves of sea buckthorn are rich in lipophilic compounds such as α-tocopherol, β-carotene and plastochromanol-8. The most common use is making tea which contains vitamins and minerals, antioxidants, amino acids and fatty acids. Tea is typically used to lower blood pressure and serum cholesterol, prevent and treat diseases of the blood vessel, and to increase immunity [4]. The stokes have common properties with berries and leaves but to a lesser extent. It is always taken as the waste of the plant. It was commonly used by the Greeks as food for horses [5]. Figure 1. Sea buckthorn distribution 9 3.1.1. Uses and benefits of sea buckthorn There are seven varieties of sea buckthorn. The most common of which are the “Hippophae rhamnoides” (common yellowthorn), and the “Hippophae salicifolia” (willow-leaf cerval). The others are not so common as follows: “Hippophae gyantsensis”, “Hippophae litangensis” and “Hippophae tibetana”. Two other species: “Hippophae goniocarpa” and “Hippophae neurocarpa”, have been described in China, but are not widely accepted as distinct species[6]. Most of the world's sea buckthorn plantations are in China. The concentration of pro-vitamins A, B2 and C is much higher than that of other fruits and vegetables such as carrots, tomatoes, oranges, etc [7]. Species Vitamin A Vitamin B1 Vitamin B2 Vitamin C Vitamin K Sea buckthorn 11 0.04 0.56 300-1600 100-200 Kiwi - - - 100-470 - Orange 0.55 0.08 0.03 50 - Tomato 0.31 0.03 0.02 11.8 - Carrot 4 0.02 0.05 8 - Table 1 Comparison of the vitamin contents of sea buckthorn and others (mg/100g) [7]. Presence of these antioxidant vitamins at high levels indicates their strong antioxidant property. The shrub serves as a warehouse for researchers in the field of biotechnology, nutraceuticals, pharmaceuticals, cosmetics and environmental sciences [7]. 3.1.2. Cosmetical usage The main use of sea buckthorn in this sector is the manufacture of an oil. The oil of sea buckthorn berry is very rich in omega 3, 6, 7 and 9 fatty acids (linolenic, linoleic, palmitoleic and oleic acids, respectively). It also contains a large amount of vitamin C (695mg/100 g), tocopherol or vitamin E (180 mg/100 g), folic acid (80 mg/100 g). It also provides carotenoids (lycopene, lutein, betacarotene and zeaxanthin), flavonoids (quercetin, isorhamnetin-3-beta-D-glucoside, isorhamnetin, kaempherol, etc) [8]. 16 - Exogenous: those obtained through a diet which are non-enzymatic such as: a) Vitamin C: facilitates electrons to free radicals helping to reduce the oxidation of cells. b) Alpha-lipoic acid (ALA): it is responsible for reducing inflammations is easier to transport to the brain than other antioxidants and facilitates the regeneration of other antioxidants such as vitamin A and C. c) Glutathione also known as master antioxidant is found in all cells of the body. d) Polyphenols: A compound found in fruits and plants. Its antioxidant action is to react to various heavy metals such as iron or copper and thus prevent the formation of free radicals in the molecule[15]  Syntetic antioxidants Synthetic antioxidants develop from the need for more effective protection than natural antioxidants, and offer a more economical option relative to natural antioxidants. Butil hydroxi-toluene (BHT) and butil hydroxi-anisol (BHA) are the most important synthetic antioxidants. Its main functions are: 1. The first function of synthetic compounds is to conserve the organoleptic properties and to preserve the nutritional quality of the food. 2. Prolong the shelf life of industrialized foods. 3. Once the compound has been intentionally introduced into the food, its function as an antioxidant does not stop the formation of radicals that are generated in oxidation[16]. 17 3.3.1.Determination of antioxidant activity by means of DPPH (2,2-diphenyl-1picrylhydrazyl) In order to know the activity of antioxidants presented in food, herbs, plants the most commonly used method is that of DPPH, which is a free radical. Free radicals are molecules that are characterized by having one or more electrons in their outermost layer condition that makes them highly reactive. Figure 5 DPPH free radical When a solution of DPPH is mixed with a substance that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present) [17]. Figure 6 Diphenyl-picryl-hydrazyl (nonradical) Representing the DPPH radical by Z´ and the donor molecule by AH. The primary reaction is: Z´ + AH = ZH + A 18 This disappeared electron causes a colour change of DPPH from its violet colour turns to pale yellow as it reacts with an antioxidant species being measured with spectrophotometer at a wavelength of 517 nm. The percentage of DPPH free radical uptake is determined by absorbance difference. It is a reaction with a kinetic of pseudo-primer order that can continue measuring the decrease in absorbance over time. The results are expressed as the value of IC50 which is defined as the concentration of the test sample that produces an inhibition of 50 % of the free radical of DPPH. It is therefore said that the IC50 value depends on the nature of the antioxidant compound and the concentration of DPPH [17]. 3.3.2. Study of the oxidative capacity of oils by Rancimat method The Rancimat is a method of measurement of the oxidative stability of oils and fats under accelerated conditions. This method is based on the induction of oxidation of the sample by exposure to high temperature and airflow. The equipment (Figure 7) has two aluminium heating blocks with electrical heating which can operate at the same or different temperatures. It has a capacity for 8 samples (4 positions per block). It admits a temperature range: from 50⁰C to 220⁰C in divisions of 1⁰C. It has an air flow capacity of 7-25 L/h[18]. The volatile oxidation products are transferred to the measuring vessel by the air stream and absorbed there in the measuring solution (distilled water). When the conductivity of this measuring solution is recorded continuously an oxidation curve is obtained whose point of inflection is known as the induction time[19]. 19 The main applications of the Rancimat method are: - Oxidation stability of fats and oils of animal and vegetable origin. - Examination of the effectiveness of antioxidants. - Oxidation stability of foods and cosmetics containing fats and oils[20] Figure 7. 743 Rancimat equipment [19] 20 4. Materials and methods 4.1. Material and chemicals The material used for the extraction was sea buckthorn stalks given by a local bio food processing company, Bio-drog-Berta Ltd., (Kalocsa, Hungary). The material contains ground particles of stalks and stems of sea buckthorn plant in the form of a small splinter it has brown colour and the smell is similar to natural horse food. Figure 8. Grinded sea buckthorn plant The following chemical were used during the experiments:  Ethanol (C2H60) supplied by Molar Chemicals Kft. Purity: 96%  Acetone (C3H60) supplied by Molar Chemicals Kft. Purity: 99.95%  Absolut ethanol (C2H60) supplied by Molar Chemicals Kft. Purity: 99.98%  Distilled water: from laboratory.  n-Pentane (C5H12) supplied by Molar Chemicals Kft. Purity: 98.03%  DPPH or 2,2-diphenyl-1-picryhydracyl, free radical (C18H12N5O6) supplied by Sigma-Aldrich Co.  Methanol (CH4O) supplied by Molar Chemicals Kft. Purity: 99.5%  Sunflower oil (Bunge Ltd, Hungary)  2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT) supplied by Fluka Chemie GmbH. Purity 99%.  terc-butil-4-hidroxianisol (BHA) supplied by Fluka chemie GmbH. Purity 98%. 21 4.2. Methods 4.2.1. Determination of moisture content First of all, the moisture content of sea buckthorn was measured. For this 3 parallel of measurements were carried out in order to obtain the highest possible accuracy. Each sample was approximately 10 g of the material. The glass without material was weighed first then the 10 g of material was introduced and weighed. The samples were kept in the oven at 105⁰C for minimum one day in order to dry the material completely (until mass constancy). After complete dryness, the sample was taken out of the oven and weighed again. The moisture content was calculated using the following formula: DC (%) =𝑀2 𝑀1 𝑥 100 Equation 2. Calculation of dry content The average of the three samples was taken. Where:  DC is the dry content of plant material (%)  𝑀1is the initial weigh of plant material before been dried in the oven  𝑀2 is the weight of plant material after been dried in the oven The moisture content can be calculated as: MC = 100-DC (%) Equation 3. Calculation of moisture content 22 4.2.2. Particle size distribution During this measurement, the particle size distribution was determined. A Retsch vibratory sieve shaker (Figure 9) was used with different sieve plate sizes (1.6; 1.4; 1; 0.8; 0.63; 0.4; <0.5mm).  The empty plates were weighted before sieving.  Sieve plates were assembled into a column with an empty tray at the bottom and with the largest diameter sieve plate at the top.  Around 85.00 g of plant material was weighted on the top of the sieve plates.  It was set for 20 minutes at 30 Hz amplitude shaking.  After that, each sieve plate was weighted back with the residue.  The percentage of the residue particles were calculated. The experiment is repeated 3 times in order to obtain the highest possible accuracy. The percentage of the different sizes was calculated by means of the formula: % = 𝑀 𝑀𝑡 ∗100 Equation 4. Calculation of percentage of different sizes on each sieve plates where Mt (g) is the total mass of material introduced into the column and M (g) is the mass of material obtained from each plate. Figure 9.3 Retsch vibratory sieve shaker 23 4.2.3. Stirred tank extraction This was the most used method in the experiments for the extraction of antioxidants from sea buckthorn stalks. The results will be compared with those of the Soxhlet method in order to determine which of the two methods is the most efficient. The following experimental design was planned to investigate the effect of temperature and H2O content in EtOH on the yield of extraction. First, 50 grams of the material was weighed and placed in a flask with a round bottom flask. Then the solvent was measured with volumetric cylinder. Ethanol-water solutions were used as extraction solvent. The proportion of ethanol was reduced as the experiments progress in such a way that one starts with 96% ethanol, 70-30% (V/V) ethanol-water, 50-50% (V/V) ethanol-water, 30-70%( V/V) ethanol-water and finally 100% distilled water. The feed solvent ratio was kept unchanged during the experiments, 50 grams of plant material in 350 ml of extraction solvent (which is 1:7 m/V ratio). The flask with the solvent and the material was placed on a support with a heating plate and a stirrer (Figure 10). The temperatures for each proportion of solvent was varied starting with 40 ºC then 60 ºC and finally 80ºC except for 96% ethanol because at 80ºC the solvent would evaporate. Agitation was kept constant in all experiments at a speed between 440 and 460 rpm approximately so that the sample was in constant motion and uniformly mixed. 24 After three hours of heating and agitation the mixture was filtered to separate the SBT content from the solution with the extract. For this purpose, vacuum filtration method was used by means of a Buchner funnel and a suction bottle. The material of interest stayed in the flask that will later be analysed to obtain the activity of the antioxidants. The dried extract in the flask was weighted. The extract was taken out from the flask and collected in a sample bottle. The extraction yield was calculated as follows: Yext. = 𝑚 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑚 𝑑𝑟𝑦 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 ∗100 [g/100 g dry material] Equation 5. Calculation of extraction yield The evaporation can be done in two different ways: by vacuum rotary evaporator (Heildolph, Germany, Figure 11 or with an automated solvent evaporation system (Biotage TurboVap) LV, Germany). The method is chosen according to the proportion of water in the solution since the water has a higher boiling point and will need a higher vacuum, temperature and longer time to evaporate. Figure 10 Stirred tank apparatus 25 Vacuum rotary evaporator The theoretical principle of the rotary evaporator is to decrease the pressure in the mixture in order to lower the boiling point of the solvent to be evaporated. The fact that it is a rotary evaporator has advantages over non-rotary evaporators: - The centrifugal and the friction force between the wall of the rotating flask and the liquid sample result in the formation of a thin film of hot solvent that spreads over a large surface and ends with the extract adhered to the wall. -The forces generated by the rotation suppress the blows, along with the above characteristics make the evaporation go smoothly and quickly. The main disadvantage of rotary evaporators is that mixtures such as ethanol and water lead to the formation of bubbles and foams that could hinder evaporation, for this must be controlled and high bath temperature and pressure to which the mixture is subjected[21]. Therefore, for certain experiments, the Biotage TurboVap was used. Figure 11. Vacuum rotary evaporator 32 The change of colour from purple to yellow of DPPH indicates that the extract has antioxidant activity and it depends on the applied concentration of it. Figure 5. Determination of IC50 value Figure 6. Colours changes of DPPH samples in the presence of sea buckthorn extracts in different concentrations 33 4.2.5.2. Study of protective effect of the extract by Rancimat method The main objective of this experiment was to compare the oxidative activities of vegetable oil samples with and without added sea buckthorn extracts obtained with Soxhlet and stirred tank extraction. In the Rancimat method the auto-oxidation of oils and fats are fasten by strong stream of air at high temperature. Volatile oxidation products cause changes in conductivity, which is monitored during the experiment. Therefore, the inflection point of oxidation curve can be measured and recorded as an induction time, that provides good characteristic value for the oxidation stability. These recorded induction times of control sunflower oil and of the sunflower oil containing the sea buckthorns extracts can be compared. If the oil samples which contain sea buckthorn extracts had longer induction time; it means that the added extract has protective effect which protects the oil from auto-oxidation. Then the extract might be used as natural preservative added to oily products. For qualification and quantification of these active compounds further studies are needed. Figure 7. Rancimat equipment 34 Figure 20. Sunflower oil For the measurement Rancimat 743 equipment was used (Figure 17) and firstly 8 test tubes were prepared. Two of them with the control sunflower oil (by weighing 3 grams of sunflower oil into a test tubes), other test tubes contain the vegetable oil + sea buckthorn extract in 1% (w/w) concentration. Three parallel measurements were carried out for each extract. Figure 8. Extracts of sea buckthorn stalks obtained with 30% EtOH and with water Sea buckthorn extracts are mixed into the vegetable oil (sunflower oil) at 1% (w/w). If the extract contains protective compounds; the oxidative stability (induction time) will increase. Once the samples were prepared in the test tubes then were assembled and placed into the previously lit Rancimat apparatus, which was preheated to a temperature of 120⁰C. Figure 9. Oil + extract 35 The measuring program was set at 120⁰C with 20 L/min air flow (default mode). As soon as all reaction and measuring tubes were assembled; the program was started and it run automatically until the induction time of each samples was recorded. After the recorded graph of conductivity versus time was obtained, copied and the recorded induction times were collected and evaluated further. From the parallel measurements average induction time was calculated with standard deviation. A characteristic graph in the presences of sunflower seed oil and oil with sea buckthorn extracts can be seen in Figure 21 and on Table 2. The measured conductivity is plotted against time and the observed induction times are spotted by triangles. Figure 21. Conductivity plotted against time with the recorded induction times of samples ID 1 Determination date Temperature Induction time automatic Sunflower oil 04.04.2019 15:14:28 120°C 2,61 h Sunflower oil 04.04.2019 15:14:29 120°C 2,61 h Oil+1% Seabuck Soxh EtOH 1 04.04.2019 15:14:31 120°C 3,02 h Oil+1% Seabuck Soxh EtOH 2 04.04.2019 15:14:32 120°C 2,89 h Oil+1% Seabuck Soxh EtOH 3 04.04.2019 15:14:34 120°C 3,06 h Oil+1% Seabuck ST1_1 04.04.2019 15:14:35 120°C 3,19 h Oil+1% Seabuck ST1_2 04.04.2019 15:14:36 120°C 3,10 h Oil+1% Seabuck ST1_3 04.04.2019 15:14:37 120°C 3,07 h Table 2. Recorded induction time by Rancimat measurement 36 5. Results & discussion 5.1. Characterization of sea buckthorn stalks As the stalks arrived to our laboratory in shredded form, and further grinding was not possible, firstly the particle size distribution of sea buckthorn stalks was evaluated. On Figure 22 the particle size distribution of the sample can be seen. Figure 22. Particle size distribution of sea buckthorn stalks Based on the results, it can be seen, that 30.7 % of the particles of the sample possessed an average size of 1 mm, while the size of 80% of the particles was between 0.8 – 1.6 mm showing a very wide particle size distribution. The moisture content of the shredded sea buckthorn was also measured. Three parallel measurements were carried out the data can be seen in Table 3. 0 5 10 15 20 25 30 35 2.5 2 1.6 1.4 1.25 1 0.8 0.63 0.4 <0.05 (%) Size(mm) Size distribution 37 1 2 3 m glass (g) 116.71 90.33 105.15 m glass+plant (g) 127.22 100.86 115.29 m plant(g) 10.51 10.53 10.14 m glass+plantdried (g) 126.46 100.14 114.57 ∆m moisture (g) 0.75 0.72 0.72 Moisture content(%) 7.23 6.84 7.1 Dry content(%) 92.77 93.16 92.9 Table 3. Moisture content of sea buckthorn stalks The average dry content of sea buckthorn stalks was 92.94 ± 0.22 %, it contained around 7% moisture. Once the dry content was obtained, it was applied to all calculation of yields of the extractions in the laboratory so the results are calculated back to dry plant material. Therefore, the obtained results can be easier comparable whether the sources of sea buckthorn are different for example by time of harvest, year, or due to geographic differences. 5.2. Results of extraction The extraction of sea buckthorn stalks was carried out using traditional Soxhlet extraction with n-pentane and with 96% ethanol solvents. Also an experimental design was carried out to investigate the effect of extraction temperature and water content in the ethanol solution on the extraction yield. For this investigation experiments at different temperatures, such as 40 – 60 and 80°C and with different water content in EtOH (4, 30, 50, 70 and 100% water contents) were carried out in stirred tank apparatus at the same stirring speed, for 3 hours of extraction time and at the same solvent to feed ration (7: 1 volume to mass ratio). During the experiments the stirring speed and the temperature of water bath was kept constant to set at the required parameters. The extraction yields were calculated as described in the Chapter 6.2.3. with Equation 5. The extraction yields are summarized in Table 4. 38 Sample No. T extraction (°C) Solvent Yield (g/ 100 g d.m.) ST1 40 96% EtOH 1.47 ST2 60 96% EtOH 1.08 ST6 40 70% EtOH 2.47 ST4 60 70% EtOH 2.81 ST5 80 70% EtOH 3.50 ST7 40 50% EtOH 2.96 ST 8 60 50% EtOH 3.15 ST 16 60 50% EtOH 4.41 ST 17 60 50% EtOH 4.55 ST 9 80 50% EtOH 3.1 ST18 80 50% EtOH 4.19 ST10 40 30% EtOH 2.77 ST11 60 30% EtOH 3.20 ST12 80 30% EtOH 3.93 ST13 40 Distilled water 1.69 ST14 60 Distilled water 1.81 ST15 80 Distilled water 1.88 SOXHLET 96% EtOH 2.93 Table 4. Extraction yields 39 Figure 23. Extraction yields The results of Soxhlet extraction is summarized in Table 5. Sample No. T extraction (°C) Solvent Yield (g/ 100 g d.m.) S.D. SOXHLET boiling point 96% EtOH 2.93 0.12 SOXHLET boiling point n-pentane 0.32 0.11 Table 5. Extraction yields of Soxhlet extraction Comparing the results of Tables 4 and 5 we come to the conclusion that the extraction yield for the same solvent with 96% ethanol the yield was almost 3 times higher than that of achieved with the stirred tank apparatus. Perhaps the extraction time was too short with the stirred tank apparatus, and the Soxhlet extraction run much longer until the plant material was fully exhausted, which could have been achieved by a stirred tank extraction in 3 hours at lower temperature. 40 60 80 0 1 2 3 4 5 96% EtOH 70% EtOH 50% EtOH 30% EtOH Distilled water Temperature (⁰C) Yield (g/ 100 g d.m.) Extraction Yields 40 60 80 40 5.2.1. Effect of temperature on the extraction yield First of all, the effect of extraction temperature on the extraction yield using different ethanolwater mixtures as extraction solvents are summarized. Figure 24. Extraction yields with 70% EtOH Figure 25. Extraction yields with 50% EtOH 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 40ºC 60ºC 80ºC Yield (%) 70% EtOH 0 1 2 3 4 5 40ºC 60ºC 80ºC Yield (%) 50% EtOH 41 Figure 26. Extraction yields with 30% EtOH Figure 27. Extraction yields with distilled water As it can be seen from the graphs (Figure 24-27), as the extraction temperature increases the extraction yield increases with them whether 70% ethanol or distilled water was used as extraction solvents. The yields were the smallest comparing them to the other yields with ethanol in the extraction solvents, as the yields increased from 1.7 to 1.9 % (g/ 100 g dry material) by increasing the temperature. Comparing all the other results there was 1.4-fold higher increased with increasing the extraction temperature from 40°C to 80°C. Using 30% EtOH the yields increased from 2.8 – 3.9 %, using 50% ethanol solvent the yields increased from 3.04.6%, and applying 70% EtOH the extraction yields increased from 2.5-3.5%, which is all 1.4-fold increase. On the other hand, this did not happen with the extraction of 50% in which the extraction yield was the highest at temperature of 60⁰C (4.04±0.77 %), but as the standard deviation of three repeated measurements show a larger deviation, the difference between the extraction yields obtained with 50% ethanol are not significant. Comparing the results, the highest extraction yield was achieved with 50% ethanol-water solution at 60⁰C. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 40ºC 60ºC 80ºC Yield (g/ 100 g d.m.) 30% EtOH 0.0 0.5 1.0 1.5 2.0 0% 40ºC 0% 60ºC 0% 80ºC Yield (g/ 100 g d.m.) Distilled water 48 Sample No. T extraction (°C) Solvent Oxidation time (h) S.D. oil - - 2.54 0.15 ST1 40 96% EtOH 3.12 0.06 ST2 60 96% EtOH 2.63 0.14 ST6 40 70% EtOH 2.69 0.19 ST4 60 70% EtOH 2.40 0.08 ST5 80 70% EtOH 2.84 0.06 ST7 40 50% EtOH 2.67 0.17 ST8 60 50% EtOH 2.69 0.08 ST9 80 50% EtOH 2.67 0.04 ST10 40 30% EtOH 2.54 0.10 ST11 60 30% EtOH 2.55 0.15 ST12 80 30% EtOH 2.51 0.02 ST13 40 Distilled water 2.48 0.07 ST14 60 Distilled water 2.39 0.11 ST15 80 Distilled water 2.48 0.03 SOXHLET at boiling point 96% EtOH 2.99 0.09 Table 8. Induction time (h) of sea buckthorn extract mixed with vegetable oil 49 Figure 33. Induction times of sunflower seed oil and sea buckthorn containing oils. At first glance, the results look very similar. The induction time of control sunflower seed oil was 2.5 ± 0.2 hours. When extract obtained with Soxhlet extraction using 96% ethanol added to the oil the induction time increased to 3.0± 0.1 h, which is a 1.2-fold increase compared it to the induction time of control oil. In the presence of extract obtained also with 96% ethanol but with stirred tank extraction at low temperature (40°C) the induction time increased to 3.1± 0.1 h, which is the highest and also shows a 1.2-fold increase. Based on these findings, sea buckthorn extracts obtained with 96% ethanol has a slight protective effect when mixed with vegetable oil in 1% concentration resulting a 1.2-fold increase in stability. It can be also stated that the compounds which are responsible for this activity are not heat-sensible but their solubilities are the highest in water-free extraction solvent. 0 0.5 1 1.5 2 2.5 3 3.5 Induction time (h) 50 6. Conclusion The following conclusions have been drawn from the study carried out: First of all, the size of the sea buckthorn stalks was evaluated. About 80% of the particles had a size between 0.8 mm and 1.6 mm. Being the size of 1mm the most common with 30.7%. The dry content of sea buckthorn stalks was 92.94 ± 0.22 %, it contained around 7% moisture. With the two methods that were used for the extraction of antioxidants from the plant sea buckthorn can be said to be almost 3 times higher extraction yield obtained by the Soxhlet method (2.93 ± 0.12) than the stirred tank at 96% EtOH and 40 ºC (1.47 g/ 100 g d.m.). Perhaps it is because the temperature and duration of the stirred tank extraction are not as high as during the Soxhlet extraction. As the extraction temperature increases, the extraction yield increases with them, whether 96% ethanol or distilled water has been used as extraction solvents. The yields with distilled water were the lowest compared to the yields with ethanol, as these increased from 1.7 to 1.9 % (g/ 100 g dry matter) as the temperature increased. Comparing all the other results, there was a 1.4-fold increase as the extraction temperature increased from 40°C to 80°C. On the other hand, this did not occur with the 50% extraction where the extraction yield was the highest at 60⁰C temperature (4.04±0.77 %). With respect to the progressive addition of water to the mixture, as it increases, the extraction yield increases until it reaches the maximum with 50% EtOH at 60⁰C (4.04± 0.77 %) thereafter the extraction yield decreases as the solvent acquires a greater proportion of water. The lowest extraction yield obtained with 96% ethanol 1.1 % and it increased up to 4.04% obtained with 50% ethanol. Three experiments were carried out at the middle point of extraction design in order to obtain the highest possible accuracy, obtaining an extraction yield of 4.04± 0,77 %. The residue extracted from the first assay (ST1) with 96% EtOH at 40°C was dried in the oven and re-extracted with 96% ethanol for another 3 hours at 40°C. The residue from the first assay (ST1) with 96% EtOH at 40°C was dried in the oven and re-extracted with 96% ethanol for another 3 hours at 40°C. The extraction yield was 1.0% at this stage. The same steps were repeated again for 1 hour, achieving a yield of 0.7%. In total, the cumulative yield of the extraction was 3.14%, twice that obtained after the first phase. 51 As is well known, the lower the IC50 factor the greater the antioxidant activity of the sample, in our case, the stronger antioxidant activity was measured in the presence of extract obtained with 50% EtOH at 80ºC with an IC50 of 20.26 ±0.50 μg/ml. The extracts obtained with pure water showed the least antioxidant activities with the biggest standard deviations. A comparison of the antioxidant activity of our sea buckthorn samples with synthetic antioxidants was made using the DPPH method, BHA showed the highest antioxidant activity (7.96 μg/ml)), but the antioxidant activities of the sea buckthorn stalk extracts are also comparable with BHT antioxidant.. The induction time of the control sunflower seed oil was 2.5 ± 0.2 hours, and it increased when extract obtained with Soxhlet extraction using 96% ethanol added to the oil to 3.0± 0.1 h, which is an increase of 1.2-fold. . Based on this, the sea buckthorn extracts obtained with 96% ethanol have a slight protective effect when mixed with vegetable oil at a concentration of 1%, resulting in an increase in stability of 1.2 times. Based on these results, it can be concluded that value-added extract can be obtained from a waste, namely from sea buckthorn stalks using either 96% ethanol in Soxhlet extraction or 50% ethanol-water solution in the stirred tank apparatus. The extracts can be obtained at the highest yield (4.3%) in the stirred tank apparatus with strong antioxidant activity, which is comparable with that of synthetic antioxidant. While the extract obtained with 96% ethanol with Soxhlet extraction possessed a modest protection effect mixed it into sunflower seed oil in 1%. Further studies are required to map the individual compounds which might be responsible for these activities. 52 7. References 1. https://es.wikipedia.org/wiki/Hippophae [𝑎𝑐𝑐𝑒𝑠𝑠𝑒𝑑 27 𝑜𝑓 𝑚𝑎𝑟𝑐ℎ 2019] 2. Olas, B. Seabuckthorn as a source of important bioactive compounds in cardiovascular diseases (2016). DOI: 10.1016/j.fct.2016.09.008. 3. https://extramineral.lat/beneficios-de-espino-cerval-de-mar/ 4. https://greekgoesketo.com/2018/12/31/hippophae/ [𝑎𝑐𝑐𝑒𝑠𝑠𝑒𝑑 10 𝑜𝑓 𝑚𝑎𝑦 2019] 5. Rajchal, R. Seabuckthorn (Hippophae salicifolia) Management Guide 2009 https://www.rufford.org/files/04.05.06%20Manual%20Guide_0.pdf 6. G. Korekara , T. Stobdana , H. Singhb , O.P. Chaurasiaa and S.B. Sing. Phenolic content and antioxidant capacity of various solvent extracts from seabuckthorn (Hippophae rhamnoides L.) fruit pulp, seeds, leaves and stem bark. 2011 DOI: 10.1556/AAlim.40.2011.4.4 7. Gateway International Inc. Seabuckthorn and Health Summary 2009 8. https://www.lineaysalud.com/salud/medicinas-alternativas/aceite-de-espino-amarillo [𝑎𝑐𝑐𝑒𝑠𝑠𝑒𝑑 28 𝑜𝑓 𝑚𝑎𝑟𝑐ℎ 2019] 9. C. John Geankoplis.Transport Processes & Separation Process Principles (Includes Unit Operations) 2014 10. https://www.healthline.com/nutrition/sea-buckthorn-oil [𝑎𝑐𝑐𝑒𝑠𝑠𝑒𝑑 29 𝑜𝑓 𝑚𝑎𝑟𝑐ℎ 2019] 11. Universidad de Granada. EXTRACCIÓN SÓLIDO-LÍQUIDO Y RECUPERACIÓN DEL DISOLVENTE: OBTENCIÓN DE ACEITE DE GIRASOL 12. F.Cunill, M. Iborra. Universitat de Barcelona. Reactores Químicos 2010. 13. https://en.wikipedia.org/wiki/Soxhlet_extractor [𝑎𝑐𝑐𝑒𝑠𝑠𝑒𝑑 4 𝑜𝑓 𝑎𝑝𝑟𝑖𝑙 2019]. 14. https://www.portalantioxidantes. com/antioxidantes/[accessed 7 of april 2019]. 15. https://www.cebanatural. com/tipos − antioxidantes − segun − procedencia − propiedades − clasificacion − blog − 337. htm[accessed 8 of april 2019]. 16. http://depa. fquim. unam. mx/amyd/archivero/Seminario − Antioxidantesartificiales_26883.pdf[accessed 8 of april 2019] 17. P.Molyneux. 2004, the use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol., 26(2): 211-219. 18. 743 Rancimat, Metrohm, 1999. Page 2. 19. 743 Rancimat, Metrohm, 1999. Page 59. 53 20. Dr LM. Paucar Menacho.Estabilidad a la oxidación de aceites y grasas por el método rancimat. 21. https://labsociety.com/lab-equipment-category/rotary-evaporators/ [𝑎𝑐𝑐𝑒𝑠𝑠𝑒𝑑 12 𝑜𝑓 𝑎𝑝𝑟𝑖𝑙 2019].