Mircowave-Assisted Extraction of Phytochemical Constituents in Roselle (Hibiscus Sabdariffa L.)

Journal of Pharmaceutical Research International

32(2): 1-12, 2020; Article no.JPRI.55233 ISSN: 2456-9119 (Past name: British Journal of Pharmaceutical Research, Past ISSN: 2231-2919, NLM ID: 101631759)

Mircowave-Assisted Extraction of Phytochemical Constituents in Roselle (Hibiscus sabdariffa L.)

Minh Phuoc Nguyen1*

1Faculty of Biotechnology, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam.

Author’s contribution

The sole author designed, analysed, interpreted and prepared the manuscript.

Article Information

DOI: 10.9734/JPRI/2020/v32i230397 Editor(s): (1) Dr. Vasudevan Mani, College of Pharmacy, Qassim University, Buraidah, Kingdom of Saudi Arabia. Reviewers: (1) Ochieng O. Anthony, Sumait University, Tanzania. (2) Md. Afjalus SIraj, University of Hawaiʻi at Hilo, USA. Complete Peer review History: http://www.sdiarticle4.com/review-history/55233

Received 20 December 2019 Accepted 27 February 2020 Original Research Article Published 04 March 2020

ABSTRACT

Roselle (Hibiscus sabdariffa L.) is natural source of valuable components such as phenolics, flavonoids and anthocyanins. Anthocyanin is normally used in the food technology to replace synthetic pigments as well as medicinal properties due to antioxidants. Microwave irradiation is a feasible green solvent extraction method receiving great attention as it utilizes solvent at elevated temperature and controlled pressure conditions. Microwave extraction emerges as a promising inexpensive, simple and efficient technique. In our research, various variable like microwave power (20, 40, 60, 80, 100 W), microwave frequency (10, 20, 30, 40, 50 GHz), the microwave irradiation duration (5, 10, 15, 20, 25 min), liquid to solid ratio (water: material, 2:1, 3:1, 4:1, 5:1, 6:1), extraction temperature (50, 55, 60, 65, 70C), number of extraction cycles (1, 2, 3, 4, 5) influencing to the microwave-assisted extraction of phytochemicals in roselle (Hibiscus sabdariffa L.) calyx were thoroughly examined. Total total phenolic (mg GAE/ 100 g), total flavonoid (mg GE/ 100 g), anthocyanin (mg/100 g) were key indicators to define the optimal variable. Our results revealed that microwave power at 80 W, frequency 40 GHz, duration 15 min, liquid to solid ratio 4:1, temperature 55C, 4 cycles of extraction were appropriate for extraction of phytochemical components inside roselle (Hibiscus sabdariffa L.) calyx.

Keywords: Roselle calyx; Hibiscus sabdariffa L.; microwave extraction; phenolic; flavonoid; anthocyanin. ______

*Corresponding author: E-mail: [email protected];

Nguyen; JPRI, 32(2): 1-12, 2020; Article no.JPRI.55233

1. INTRODUCTION as colouring agent in pharmaceutical products [27,28]. It has been utilized to cure different Roselle (Hibiscus sabdariffa L.) is an ideal crop degenerative diseases like hypertension, for developing countries. Rain or high humidity hyperlipidemia, cancer and other inflammatory during the harvest time and drying process can diseases of liver and kidney [29]. It’s also proven downgrade the quality of the calyces and reduce to treat cardiac, diuretic, sore throat and cough, the yield. It can be used as food and fibre [1]. cholerectic, febrifugal and hypotensive effect, Roselle contains protein, fat, carbohydrate, fiber, liver disorder, decrease the viscosity of the and mineral, vitamin. The chemical components blood, induce lactation and stimulate intestinal contained in Hibiscus sabdariffa calyx include peristalsis [30-33]. anthocyanins, flavonoids, polyphenols polysaccharides and organic acids having Microwave-extracted extraction is a method that different modern therapeutic applications [2-6]. utilizes a solvent to extract the phytochemical Phenolic derivatives and flavonoids natural components from herbs. In this process, the compounds in plants and paly several roles in improved extraction happens as s result of the plant’s life such as general growth, change in the herbal cell texture created by reproduction, and defence against parasites and electromagnetic waves [34]. Microwaves are pests [7]. Flavonoids themselves are a group of electromagnetic radiations having frequency from hydroxylated phenolic compounds having a 0.3 to 300 GHz. The combination of thermal and benzo-γ-pyrone structure and are ubiquitously mass gradients contribute to the high yield and occurring in plants [8]. Anthocyanins are steroid short extraction duration of micro-extracted or triterpenoid glycosides, common in a large extraction. The variables that affect the extraction number of herbs. According to the structure of recovery of this extraction process are the power the aglycone or sapogenin, anthocyanins are and frequency of the microwaves, the duration of classified as neutral and acid type, the so-called the microwave irradiation, the moisture content neutral anthocyanins are derivatives of steroids and particle sizes of the herbs, the kind and with spiroketal side chains which are almost concentration of the solvent, the ratio of solid to exclusively present in the monocotyledonous liquid, the extraction temperature, the extraction angiosperms and the acid anthocyanins that pressure, and the number of extraction cylces possess triterpenoid structure type, which is the [35]. Microwave-assisted extraction (MAE) has most common and occur mainly in the been recognized as a technique with several dicotyledonous angiosperms. It has effect on advantages over other extraction methods, such cold blooded animals, also to have the analgesic, as reduction of costs, extraction time, energy anti-nociceptive, antioxidant activity, to impair the consumption, and CO2 emissions. However, digestion of protein, to cause hypoglycemia and there was not many research mentioned to the to act as antifungal and antiviral agents [9]. The application of microwave-assisted extraction to amount of L-ascorbic acid extracted was 83.1 extract phytochemicals in roselle. Maceration mg/100 g [10]. Hibiscus sabdariffa L. calyx and ultrasound-assisted techniques were contained polyphenols of the flavonol and compared in assessing the extraction flavanol type in simple or polymerised form [11]. performance of anthocyanin in roselle [36]. Extracts of dried calyces were able to decrease Objective of our study focused on various low-density lipoprotein cholesterol, triglyceride, variables such as microwave power, microwave total cholesterol and lipid peroxidaxion [12-14]. frequency, the microwave irradiation duration, According to one report, the total phenolic liquid to solid ratio, extraction temperature, content was found to be 41.07 mg GAE/g [15]. number of extraction cycles affecting to the According to another research, roselle had total microwave-assisted extraction of phytochemicals phenols 29.178 mg/kg [16]. Meanwhile, flavonoid in roselle (Hibiscus sabdariffa L.) calyx. was observed in calyx extracts 148.35 mg/g [17]. Fresh or dried calyces of Hibiscus sabdariffa L. 2. MATERIALS AND METHODS are utilized to convert into herbal tea, hot and cold beverage, fermented drink, soft drink, wine, 2.1 Materials jam, jellied confectionary, sauce, marmalade, ice cream, chocolate, flavouring agent, pudding and Roselle (Hibiscus sabdariffa L.) calyxs were cake [18-26]. Aqueous extracts of roselle naturally collected from Hau Giang province, (Hibiscus sabdariffa L.) calyces have Vietnam. After collecting, they must be kept in characteristic intense red colouration due to the dry cool box and quickly conveyed to laboratory presence of anthocyanins which could be utilised for experiments. They were subjected to washing

Nguyen; JPRI, 32(2): 1-12, 2020; Article no.JPRI.55233

and treatment. These calyxs were treated by key indicators so they were chosen to estimate different parameters such as microwave power the optimal liquid to solid ratio. (20, 40, 60, 80, 100 W), microwave frequency (10, 20, 30, 40, 50 GHz), the microwave 2.2.5 Effect of extraction temperature (C) in irradiation duration (5, 10, 15, 20, 25 min), liquid phytochemical extraction of Hibiscus to solid ratio (water: material, 2:1, 3:1, 4:1, 5:1, sabdariffa L. calyx 6:1), extraction temperature (50, 55, 60, 65, 70C), number of extraction cycles (1, 2, 3, 4, 5). Raw Hibiscus sabdariffa L. calyx was extracted At the end each treatment, samples were by different extraction temperature values (50, analyzed total phenolic (mg GAE/ 100 g), 55, 60, 65, 70C). Total phenolic (mg GAE/100 flavonoid (mg GE/ 100 g), anthocyanin (mg/100 g), total flavonoid (mg GE/ 100 g), total g) content to define the optimal value. anthocyanin (mg/100 g) were major indicators so they were chosen to identify the optimal 2.2 Researching Methods extraction temperature.

2.2.1 Effect of microwave power in phytochemical extraction of Hibiscus 2.2.6 Effect of the number of extraction cycles sabdariffa L. calyx in phytochemical extraction of Hibiscus sabdariffa L. calyx Raw Hibiscus sabdariffa L. calyx was extracted by various microwave power values (20, 40, 60, Raw Hibiscus sabdariffa L. calyx was extracted 80, 100 W). Total phenolic (mg GAE/100 g), total by different numbers of extraction cycles (1, 2, 3, flavonoid (mg GE/ 100 g), total anthocyanin 4, 5). Total phenolic (mg GAE/100 g), total (mg/100 g) were key indicators so they were flavonoid (mg GE/ 100 g), total anthocyanin chosen to define the optimal microwave power. (mg/100 g) were important indicators so they were chosen to identify the optimal number of 2.2.2 Effect of microwave frequency in extraction cycles. phytochemical extraction of Hibiscus sabdariffa L. calyx 2.3 Chemical and Statistical Analysis

Raw Hibiscus sabdariffa L. calyx was extracted by different microwave frequency values (10, 20, Total polyphenol content (mg GAE/100 g) was 30, 40, 50 GHz). Total phenolic (mg GAE/100 g), determined by FolinCiocalteu reagent method total flavonoid (mg GE/ 100 g), total anthocyanin [37]. Aluminum chloride colorimetric method was (mg/100 g) were major indicators so they were used for flavonoids (mg QE/100 g) determination chosen to determine the optimal microwave [38]. Total anthocyanin content (mg) was frequency. quantified by spectrophotometry [39]. The experiments were run in triplicate with three 2.2.3 Effect of microwave irradiation duration different lots of samples. Statistical analysis was in phytochemical extraction of Hibiscus performed by the Statgraphics Centurion XVI. sabdariffa L. calyx 3. RESULTS AND DISCUSSION Raw Hibiscus sabdariffa L. calyx was extracted by different microwave irradiation duration values 3.1 Effect of Microwave Power (W) in (5, 10, 15, 20, 25 min). Total phenolic (mg Phytochemical Extraction of Hibiscus GAE/100 g), total flavonoid (mg GE/ 100 g), total sabdariffa L. Calyx anthocyanin (mg/100 g) were main indicators so they were chosen to select the optimal Extract of roselle calyces has shown the microwave irradiation duration. presence of biochemicals such as anthocyanins, 2.2.4 Effect of liquid to solid ratio (water: protocatechuic acid, flavonoids, and anthocyanin Hibiscus sabdariffa L. calyx) in [12,40-43]. According to one report, Roselle phytochemical extraction calyces contained ascorbic acid (140.13 mg/100 g), total anthocyanins (622.91 mg/100 g) and Raw Hibiscus sabdariffa L. calyx was extracted total phenolics (37.42 mg/g) [44]. Meanwhile the by different liquid to solid ratio (water: Hibiscus anthocyanin was detected at 1.8% in red roselle sabdariffa L. calyx, 2:1, 3:1, 4:1, 5:1, 6:1). Total calyx [45]. Polyphenols in roselle included phenolic (mg GAE/100 g), total flavonoid (mg delphinidin and cyanidin having antioxidant GE/ 100 g), total anthocyanin (mg/100 g) were activities beneficial for human body [46-49].

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Aqueous extracts of roselle (Hibiscus sabdariffa 3.3 Effect of Microwave Irradiation L.) calyces have characteristic intense red Duration (min) in Phytochemical colouration due to the presence of anthocyanins, Extraction of Hibiscus sabdariffa L. which are flavonoids are water-soluble natural Calyx pigments [50]. In our research, raw Hibiscus sabdariffa L. calyx was extracted by different The microwave power and irradiation times microwave power values (20, 40, 60, 80, 100 W). influence each other to a great extent [53]. In our Total phenolic (mg GAE/100 g), total flavonoid research, raw Hibiscus sabdariffa L. calyx was (mg GE/ 100 g), total anthocyanin (mg/100g) extracted by different microwave irradiation were important indicators so they were chosen to duration values (5, 10, 15, 20, 25 min). Total identify the optimal microwave power. Our result phenolic (mg GAE/100 g), total flavonoid (mg showed that when microwave power increased GE/ 100 g), total anthocyanin (mg/100 g) were from 20 W to 80 W, the total phenolic, flavonoid key variables to identify the optimal microwave and anthocyanin extracted from Hibiscus irradiation duration. Our result showed that when sabdariffa L. calyx increased. There was no microwave irradiation duration increased from 5 significant difference at microwave power 80 W minutes to 15 minutes, the total phenolic, and 100 W so the optimal microwave power flavonoid and anthocyanin extracted from should be 80 W to maintain the highest content Hibiscus sabdariffa L. calyx increased also. of total phenolic, flavonoid and anthocyanin. In However if microwave irradiation duration one report, variable of power had no important extended over 15 minutes, these phytochemical effects on the yield of flavonoids [51]. One constituents decreased gradually. So the optimal studied microwave extraction of cardamom. irradiation duration should be 15 min to extract When the glands were subjected to more severe the highest content of total phenolic, flavonoid thermal stresses and localized high pressures, and anthocyanin. Total phenolic content was pressure build-up happens within the glands extracted from aromatic plants such as which results in accelerated capacity for Rosmarinus officinalis using microwave-assisted expansion and leading to cell rupture more extraction. Compared with traditional reflux rapidly than in traditional extraction [52]. extraction, microwave-assisted extraction reduced extraction duration, limited solvent 3.2 Effect of Microwave Frequency (GHz) volume and increased extraction yield of total in Phytochemical Extraction of phenolics [55]. The yield of analyte extracted can Hibiscus sabdariffa L. calyx be enhanced with an accelerate in the extraction time; however, there is a related risk of Microwaves are non-ionizing electromagnetic degradation of sensitive components [56]. One waves of frequency between 300 MHz to 300 study confirmed that extraction duration in GHz or between wavelengths of 1 cm and 1m microwave assisted process was found to [53]. Microwave energy is transfered directly to decrease with increase in temperature. This the herbal tissue through molecular interaction decrease could be realized to the fact that with the electromagnetic field via conversions of increase in temperature, the vapour pressure of electromagnetic energy into thermal energy water present inside the celery seeds increased [35,54]. In our research, raw Hibiscus sabdariffa leading to leaching out and evaporation of L. calyx was extracted by different microwave volatile oil along with moisture [57]. Extended frequency values (10, 20, 30, 40, 50 GHz). Total extraction durations increased the yield of total phenolic (mg GAE/100 g), total flavonoid (mg flavonoid, while progressively decreased GE/ 100 g), total anthocyanin (mg/100 g) were flavonoids and antiradical power [58]. main indicators so they were chosen to define the optimal microwave frequency. Our result 3.4 Effect of Ratio of Liquid to Solid showed that when microwave frequency (Water: Hibiscus sabdariffa L. Calyx) increased from 10 GHz to 40 GHz, the total in Phytochemical Extraction phenolic, flavonoid and anthocyanin extracted from Hibiscus sabdariffa L. calyx increased also. Another critical factor in microwave-assisted There was no significant difference at microwave extraction is the ratio of the solid herbs to the frequency 40 GHz and 50 GHz so the optimal amount of solvent. The bioactive ingredients in microwave frequency should be conducted at 40 the herbs can effectively dissolve when large GHz to preserve the highest content of total amounts of solvent are used, thereby leading to phenolic, flavonoid and anthocyanin. improved extraction yields. Solvent specification

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is the most vital variable because the solvent optimum conditions for fresh roselle calyxes, affects the absorbance of the microwave energy, fresh calyxes to water ratio was 1:2. For dried as determined by the dissipation factor [59,60]. roselle calyxes, the optimum conditions were The solvent must have an affinity for the target 1:10 ratio of dried calyxes to water. The total component and an capability to absorb anthocyanin, total phenolic contents in fresh microwave energy [61]. In a higher contact roselle calyxes were 37.67±0.02 mg/100 g, surface area, the extraction efficiency rises. 31.26±0.75 mg gallic acid/g. The total Similarly, finer particles permit enhanced anthocyanin, total phenolic contents in dried penetration of the microwave. Fine particles may roselle calyxes were 340.97±0.15 mg/100 g, stance some technical difficulties; filtration is 31.18± 0.62 mg gallic acid/g [62]. Anthocyanin applied to prepare the matrix. In our research, from roselle has been previously extracted using raw Hibiscus sabdariffa L. calyx was extracted by various solvents such as water [63-68], methanol different liquid to solid ratios (water: Hibiscus [63], ethanol acidified by HCl [69] and in the sabdariffa L. calyx, 2:1, 3:1, 4:1, 5:1, 6:1). Total instant pressure drop system solvent [70]. A high phenolic (mg GAE/100 g), total flavonoid (mg solvent ratio with microwave-assisted extraction GE/ 100 g), total anthocyanin (mg/100 g) were showed consistent results [71,72]. When large major variables to identify the optimal liquid to quantities of solvent were utilized, the extraction solid ratio. Our result showed that when liquid to yield initially increased and then decreased as solid ratio increased, the phytochemical contents the solid-to-liquid ratio decreased [72,73]. When decreased afterward. The optimal liquid to solid the amount of solvent was reduced (high-solid ratio should be 4:1 to receive the highest content herbal materials), microwave energy may have of total phenolic, flavonoid and anthocyanin. In been absorbed and dispersed by the large another report, the effect of solvent types and amount of plant materials [71], thereby solute to solvent ratios were also evaluated by increasing the solid mass, and decreasing the maceration and ultrasound-assisted to extract surface area available for solvent to penetrate anthocyanin. Water was a better choice of the plant materials and solubilize the target solvent as compared to ethanol [36]. The molecules [74].

Table 1. Effect of microwave power to the total phenolic, flavonoid and anthocyanin extracted from Hibiscus sabdariffa L. calyx

Microwave power (W) 20 40 60 80 100 Total phenolic 51.38±0.03c 54.04±0.00b 55.17±0.02ab 56.83±0.03a 56.90±0.01a (mg GAE/ 100 g) Total flavonoid 10.27±0.01c 13.54±0.00b 13.91±0.01ab 14.23±0.00a 14.26±0.02a (mg GE/ 100 g) Anthocyanin 18.53±0.02c 20.97±0.01b 21.29±0.00ab 21.86±0.02a 21.90±0.03a (mg/100 g) Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%)

Table 2. Effect of microwave frequency (GHz) to the total phenolic, flavonoid and anthocyanin extracted from Hibiscus sabdariffa L. calyx

Microwave 10 20 30 40 50 frequency (GHz) Total phenolic 56.83±0.03c 57.41±0.00b 58.63±0.03ab 59.52±0.02a 59.59±0.03a (mg GAE/ 100 g) Total flavonoid 14.23±0.00b 14.45±0.01ab 14.68±0.00ab 14.87±0.01a 14.90±0.01a (mg GE/ 100 g) Anthocyanin 21.86±0.02b 22.14±0.01ab 22.59±0.02ab 22.84±0.03a 22.87±0.00a (mg/100 g) Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%)

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Table 3. Effect of microwave irradiation duration (min) to the total phenolic, flavonoid and anthocyanin extracted from Hibiscus sabdariffa L. calyx

Microwave irradiation 5 10 15 20 25 duration (min) Total phenolic 59.52±0.02c 64.29±0.00b 67.15±0.03a 66.49±0.02ab 62.37±0.00bc (mg GAE/ 100 g) Total flavonoid 14.87±0.01d 17.75±0.03ab 18.62±0.02a 18.25±0.01ab 17.13±0.02b (mg GE/ 100 g) Anthocyanin 22.84±0.03c 23.27±0.01bc 24.01±0.00a 23.81±0.01ab 23.54±0.00b (mg/100 g) Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%)

Table 4. Effect of liquid to solid ratio to the total phenolic, flavonoid and anthocyanin extracted from Hibiscus sabdariffa L. calyx

Liquid to solid ratio 2:1 3:1 4:1 5:1 6:1 Total phenolic 67.15±0.03a 57.25±0.00ab 50.49±0.03b 29.42±0.00c 11.63±0.02d (mg GAE/ 100 g) Total flavonoid 18.62±0.02a 14.34±0.03ab 12.18±0.01b 8.33±0.03c 5.47±0.01d (mg GE/ 100 g) Anthocyanin 24.01±0.00a 21.84±0.01ab 18.53±0.02b 16.72±0.01bc 14.85±0.03c (mg/100 g) Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%)

3.5 Effect of Extraction Temperature (oC) GAE/100 g), total flavonoid (mg GE/ 100 g), total in Phytochemical Extraction of anthocyanin (mg/100 g) were important Hibiscus sabdariffa L. Calyx indicators so they were chosen to identify the optimal extraction temperature. Our result High-temperature extraction can be gainful with showed that accelerated temperature from 50C the resulting increase in solubility. This is to 70C can hampered the phytochemicals. because higher temperature causes increased There was not significant difference at extraction intermolecular interactions within the solvent, temperature 50C and 55C. Therefore the giving increase to higher molecular motion which optimal extraction temperature should be 55C to rises the solubility. The accelerating temperature receive the highest content of total phenolic, may cause a cellular pressure build up which flavonoid and anthocyanin to save duration of may create cell rupture and opening of the cell extraction. The optimum conditions for fresh and matrix, and as a result, increased elements dried roselle calyxes, the extraction temperature accessibility to be extracted into the solution. of 50oC for 30 min [62]. Dried roselle calyces at a When the temperature of water is raised, there is ratio of 1:10 (dried roselle calyces: water) were a steady decrease in its permittivity, viscosity and extracted at 50C for 30 minutes [32]. surface tension but an increase in its diffusivity characteristics. The increased temperature can 3.6 Effect of the Number of Extraction overcome the solute–matrix interaction caused Cycles in Phytochemical Extraction of by van der Waals forces, hydrogen bonding, Hibiscus sabdariffa L. Calyx dipole attraction of the solutes molecules and active sites in the matrix [75]. However, thermally The outcome of stirring is related to the mass labile compounds are degraded at elevated transfer manner in the solvent phase. Therefore, temperatures. Sufficient temperature is balance between the aqueous and vapor phases neccessary to optimize the extraction efficiency, can be achieved more rapidly. The use of avoid thermal degradation of the target analytes, agitation in MAE quickens the extraction by and to supply reproducible processing conditions enhancing desorption and dissolution of active [48,53]. Raw Hibiscus sabdariffa L. calyx was compounds bound to the sample matrix. Through extracted by different extraction temperature stirring, the disadvantages of the use of low values (50, 55, 60, 65, 70C). Total phenolic (mg solvent-tosolid ratio can be reduced, together

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Table 5. Effect of extraction temperature (C) to the total phenolic, flavonoid and anthocyanin extracted from Hibiscus sabdariffa L. calyx

Extraction 50 55 60 65 70 temperature (oC) Total phenolic 52.84±0.02a 52.27±0.00ab 51.89±0.03b 51.23±0.02bc 50.49±0.03c (mg GAE/ 100 g) Total flavonoid 13.20±0.03a 13.17±0.02a 12.98±0.01ab 12.79±0.00ab 12.18±0.01b (mg GE/ 100 g) Anthocyanin 19.07±0.00a 19.03±0.01a 18.94±0.02ab 18.79±0.01ab 18.53±0.02b (mg/100 g) Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%)

Table 6. Effect of the number of extraction cycles to the total phenolic, flavonoid and anthocyanin extracted from Hibiscus sabdariffa L. calyx

Number of extraction 1 2 3 4 5 cycles Total phenolic 52.27±0.00b 52.68±0.02ab 52.89±0.03ab 52.97±0.00a 53.00±0.02a (mg GAE/ 100 g) Total flavonoid 13.17±0.02b 13.58±0.03ab 13.84±0.01ab 13.96±0.03a 13.99±0.03a (mg GE/ 100 g) Anthocyanin 19.03±0.01c 19.86±0.00bc 20.24±0.02b 20.57±0.00ab 20.72±0.01a (mg/100 g) Note: the values were expressed as the mean of three repetitions; the same characters (denoted above), the difference between them was not significant (α = 5%) with the minimization of the mass transfer barrier 4. CONCLUSION created by the concentrated solute in a localized region resulting from insufficient solvent. It is Roselle (Hibiscus sabdariffa) is a good source of possible to observe the difference between phytochemical constituents such as dietary suspensions with and without stirring [76]. The antioxidant, phenolic, ascorbic acid, carotenoid microwave cycle used must be carefully providing high antioxidant activity with potential monitored, because microwave-assisted health benefits. The application of microwave extraction offers quick release of the target extraction of functional constituents from roselle components in the surrounding extraction solvent calyx results in effective manner. Electro- and longer extraction durations could accelerate magnetic waves are indeed absorbed selectively the decomposition of extracted phenolics for by media possessing a high dielectric constant extended extraction under these harsh conditions resulting in more effective heating. In this [77]. In our research, raw Hibiscus sabdariffa L. research, we have successfully identified major calyx was extracted by different numbers of variables influencing to the phytochemical extraction cycles (1, 2, 3, 4, 5). Total phenolic extraction inside roselle calyx under microwave. (mg GAE/100 g), total flavonoid (mg GE/ 100 g), total anthocyanin (mg/100 g) were important CONSENT AND ETHICAL APPROVAL indicators so they were chosen to identify the optimal number of extraction cycles. Our result It is not applicable. showed that when the number of extraction COMPETING INTERESTS cycles increased from 1 to 4 cycles, the phytochemical stability increased respectively. Author has declared that no competing interests However there was not significant difference if exist. we prolonged the extraction cycle over 4 units. Therefore the optimal number of extraction REFERENCES cycles should be four to receive the highest content of total phenolic, flavonoid and 1. Inês Da-Costa-Rocha, Bernd Bonnlaender, anthocyanin. Hartwig Sievers, Ivo Pischel, Michael

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