Phytochemical screening and total phenolics and flavonoids contents of hibiscus rosa sinensis. L cultivated in Viet Nam

MedPharmRes, 2017, 152 MedPharmRes journal of University of Medicine and Pharmacy at Ho Chi Minh City homepage: and Original article Phytochemical Screening and Total Phenolics and Flavonoids Contents of Hibiscus Rosa Sinensis. L Cultivated in Viet Nam Truong Thi Han, Le Thi Ly*, Tran Thi Minh School of Biotechnology, International University HCM city- Vietnam National University HCMC. Received September 23, 2017: Accepted October 12, 2017: Published online December 10, 2017 Abstract: content (TFC) and Fourier-transform infrared spectroscopy (FTIR) spectral analysis of Hibiscus Rosa Sinensis L. extracts (crude extract, n-hexane extract, ethyl acetate extract, and methanol extract) for further applications in pharmaceutical development. Method: Total phenolics were estimated by Folin-Ciocalteu method; while, Aluminum Hibiscus Rosa Sinensis compound was determined using a FTIR-spectrophotometer. Result: Results showed the leaves extracts to extract, ethyl acetate extract and n-hexane extract were 57.09 ± 0.35 mg/g, 70.98 ± 0.03 mg/g, 21.31 ± 0.01 mg/g, and 18.45 ± 0.003 mg/g as gallic acid equivalent, respectively. Crude extract, methanol extract, ethyl acetate extract and mg/g as rutin equivalent. FTIR spectra of four extracts were comparable and showed the presence of nitro compounds and ring aromatic compounds. Conclusion: Our results indicate the potential of exploiting Hibiscus Rosa Sinensis leaves as a source of chemotherapeutic compounds, and it is worthy doing further researches on isolated bioactive compounds for developing novel functional foods or new drugs. Key words: phytochemical. 1. INTRODUCTION Herbal plants contain a great amount of chemical diversity which provides opportunities for new drug formulations either as isolated compounds or as standardizing extracts. Biological activities of plants strongly depend on its constituents especially phenolics. They encompass a very large and diverse group of aromatic compounds characterized by a benzene ring with one or more hydroxyl groups produced by plant secondary metabolites mainly a common group of polyphenolic compounds which have been reported to more than 4500 derivatives having known properties which include free radical scavenging, inhibition action and anti-corrosive agents [11]. Hibiscus Rosa Sinensis L. is a genus of an evergreen herbaceous plant (Malvaceae) commonly cultivated in all over the world. In traditional, many parts of this plant are proposed to be rich in phytonutrients contributed for aphrodisiac, bronchial catarrh, fever, strangury, cystitis and other genito-urinary troubles, alopecia, and diabetes. Leaves have been treated anodyne, emollient and aperient; gonorrhea, alopecia and also used for blackening hair; the buds reduced burning of the body, urinary discharges, seminal weakness, piles, uterine and vaginal discharges; promote the growth of the fetus; The root is valuable in coughs. (Anil Kumar and Ashatha Singh, 2012). Crude water-ethanolic extract of Hibiscus rosa sinensis leaves were exhibited excellent antioxidant activities comparing to synthetic antioxidants (BHT, BHA) (Faten et al, 2011). However, it is rarely used in Vietnam and often be confused with Hibiscus Sabdariffa. In this study we aimed to qualify Hibiscus Rosa Sinensis planted in Ho Chi Minh city. * Address correspondence to this author at the School of Biotechnology, International University Ho Chi Minh city - Vietnam National University HCMC, Ho Chi Minh city, Vietnam; E-mails: ly.le@hcmiu.edu.vn © 2017 MedPharmRes 53 2. MATERIALS AND METHOD The fresh leaves of Hibiscus rosa-sinensis were collected from Ho Chi Minh National University campus in Ho Chi Minh city, Vietnam in October 2016. Fresh leaves with uniform structure were chosen and washed with distilled water. After that, samples were air dried till constant weight and then ground to powder. The samples were stored in air tight bags for further periodical uses. Chemicals and reagents The chemicals used include absolute ethanol, n-hexane, ethyl acetate (EtOAc), methanol (MeOH) purchased from Chemsol Company, Viet Nam. Thin layer chromatography (TLC), aluminum chloride (AlCl3), and sodium carbonate (Na2CO3) were purchased from MERCK, Germany. Folin- Ciocalteu reagent, gallic acid were products of Sigma- Aldrich (St. Louis, MO, USA). Preparation of Hibiscus rosa-sinensis extract The plant powder was extracted by 15L of 96% ethanol under room temperature for 1 week. Then, the aqueous the residue was added by fresh ethanol. This process was repeated three times until a clear colourless solution was obtained. The crude extract was concentrated under reduced pressure at 60°C, using a rotary evaporator. Then, the crude extract was soaked by suitable solvent (n-hexane, ethyl acetate, and methanol) to get fraction extractions. Dried extracts were then weighed and stored in a refrigerator. The percentage yield of extract was calculated using the expression. Yield (%) = Amount of dried extract/wt. of sample ×100 Preliminary phytochemical screening A portion of crude extract was used for phytochemical tannins, steroids, terpenoids using standard procedure (Harborne, 1984; Trease and Evans 2002). Determination of total phenolics content (TPC) The total phenolics content was determined according to the Folin-Ciocalteu method [17]. The extracts and the standard gallic acid at different concentrations were taken in test tubes and 1.5 mL of FC reagent (10%; w/v) was added, after 5 minutes add to the mixture 1.5mL of sodium carbonate (20%; w/v). After incubation for 5 min, the mixtures were allowed to stand for 2 hours under dark condition. The absorbance was measured at 750 nm in a spectrophotometer. The concentration of total phenolics is expressed in terms of gallic acid equivalence (mg GAE/g). H. Rosa extract was determined by the aluminum chloride colorimetry assay [12]. Five milliliters extract at 0.4 mg/mL in methanol; 5 mL of 2% AlCl3 solution was added, and the mixtures were allowed to stand for 10 min. Then, the absorbance was measured at 415 nm. - ibration curve, and the result was expressed as mg rutin equivalent per g dry weight. Fourier-transform infrared spectroscopy (FTIR) analysis Fourier-transform infrared spectroscopy (FTIR) is a technique recently used to qualify the presence of bioactive compounds from a biological and pharmaceutical sample based on the determination of the interaction between an IR radiation and a sample that can be solid, liquid or gaseous. The radiation absorption of chemical functional groups response at different frequencies, so FTIR will measure the frequencies at which the sample absorbs, and the intensities of these absorptions [3]. Sampling All sample extraction and measurement system components was maintained at room temperatures that transported to the FTIR gas cell for analysis. If sample conditioning is used, then the condenser system (or another device) should minimize the contact between the condensed Determination of Gaseous Compounds by Extractive Direct Interface Fourier Transform Infrared (FTIR) Spectroscopy). Analysis transform infrared (FTIR) spectrometer gas cell. Individual radiation that is proportional to their concentration. The simultaneously. Statistical analysis The data were analyzed using Microsoft excel 2013 soft- ware and obtained mean ± SD (n=3). 3. RESULTS AND DISCUSSION Preparation of Hibiscus Rosa Sinensis extracts 10 kg of fresh Hibiscus Rosa Sinensis L. yield approximately 2 kg of dried powder, 205.77 g of crude extract, 93.58 g of n-hexane extract, 4.45 g of ethyl acetate extract, and 103.42 g of methanol extract. Each extract was measured moisture for calculating dried mass and the Preliminary phytochemical screening A preliminary phytochemical screening test is not only a simple, quick, and inexpensive procedure but also an important tool in a bioactive analysis that gives a brief summary of phytochemicals in an unknown mixture. According to the preliminary test, there were the present Alkaloids and coumarin were recorded to not detected in Hibiscus Rosa Sinensis. Phytochemical Screening and Total Phenolics and Flavonoids Contents MedPharmRes, 2017, Vol. 1, No. 1 54 These metabolites are widely used in various pharmaceutical preparations. In Asian traditional medicine, the tannin-containing plant extracts are used as astringents, against diarrhea, as diuretics, against stomach and duodenal and haemostatic pharmaceuticals [5]. Saponins also have many pharmacological properties, such as antifungal, immunostimulant, hypocholesterolemic and hypoglycemic [10]. As consequence, this plant has the great economic importance of this group of plant constituents. Table 1. Preliminary phytochemical screening of H. Rosa Sinensis Chemical Constituents (+) Positive/ (-) Negative Alkaloids - Glycosides + Steroids + Saponins + Coumarins - Flavonoids + Tannins + + refers presence and - refers absence content (TFC) Polyphenols are the most abundant and widespread components in plants. There are more than 8000 phenolics constituents which have various bioactivities which could 2015; Kuti, 2004). Flavonoids are the major subgroup of various biological functions in plants, including protection against ultraviolet (UV) radiation and phytopathogens, as a visual signal that attracts pollinators [7], as well as antioxidant components by free radicals scavenging activities [16]. Therefore, the obtaining of phenolics and process biological activities. According to previous studies, Hibiscus Rosa Sinensis [16]. In the present study, results obtained from Hibiscus (see table 2). The amount of total phenolics varied among four extracts of H. Rosa Sinensis, crude extract, methanol extract, ethyl acetate extract and n-hexane extract were 57.09 ± 0.35 mg/g, 70.98 ± 0.03 mg/g, 21.31 ± 0.01 mg/g, and 18.45 ± 0.003 mg/g as gallic acid equivalent, respectively. This result showed that the total phenolics content were relatively high in methanol extract and crude extract; while crude extract, methanol extract, and ethyl acetate extract 26.87 ± 0.01 mg/g, 21.08 ± 0.03 mg/g, 21.70 ± 0.001 mg/g in n-hexane extract was only 14.95 ± 0.02 mg/g. So, the potential of many biological activities is likely to express in these extracts compared to n-hexane. Fourier-transform infrared spectroscopy (FTIR) analysis FTIR has been widely used as a valuable tool for the groups present in a mixture of plant extracts (Sasidharan, 2011). One of the advantages of IR spectroscopy is that it has the ability to identify chemical in small quantities without isolation or derivatization, so it can eliminate deactivations and transformations during preliminary preparation. The spectrum of testing compounds can be Hydroxyl or amino groups mainly signal very characteristic -1, around 1615- 1495cm-1 are consistent with an aromatic compound; while frequencies from 1600 to 1268cm-1, indicate absorption by nitrate, ammonium, aliphatic hydrocarbons, aliphatic and aromatic carboxylic acids, amino acids and carbohydrates [2]. All the spectra of Hibiscus Rosa Sinensis were extracted in different types of organic solvents investigated shared certain spectral similarities (Figure 2). FTIR spectra for peaks:1197cm-1, 1230cm-1, 1235cm-1, 1263cm-1, 1362cm- 1, 1374cm-1, 1418cm-1, 1457cm-1, 1489cm-1, 1500cm- 1, 1555cm-1, 1616cm-1, 1635cm-1, 1652cm-1, 1669cm-1, 1683cm-1, 1700cm-1, 1717cm-1, 1733cm-1, 1751cm-1, 1820cm-1, 1830cm-1, 1867cm-1, 3320cm-1, 3350cm-1, 3486cm-1. The absorptions of extracts were over 3000cm-1, the compound was proved to be likely unstarurated (C=C or aromatic) (Coates, 2000). 1197cm-1 peak corresponds to the secondary amine group; the 1489cm-1, 1500cm-1, 1555cm-1 indicate to aromatic nitro compounds, while the 1616cm-1, 1635cm-1, 1652cm-1, 1669cm-1, 1683cm-1 indicate to Quinone or conjugated ketone or amide; the peaks having the value of 1700cm-1, 1717cm-1 represent for carboxylic acid; the sharp peaks occur at 1733cm-1 and 1751cm-1 in 1820cm-1, 1830cm-1, 1867cm-1 peaks; Imino compounds, NH strength absorbs IR light at frequencies around 3320cm- 1 and 3350cm-1; while the peak having the value of 3486cm-1 is probably heterocyclic amine, NH strength. MedPharmRes, 2017, Vol. 1, No. 1 Truong et al. 55 Figure 1. Percentage of extracts from H. Rosa moisture and productivity. Table 2. H. Rosa Sinensis. Extracts Total phenolics content (mg gallic acid equivalent /g) (mg rutin equivalent /g) Crude extract 57.09 ± 0.35 26.87 ± 0.01 Methanol extract 70.98 ± 0.03 21.08 ± 0.03 Ethyl acetate extract 21.31 ± 0.01 21.70 ± 0.001 n-hexan extract 18.45 ± 0.003 14.95 ± 0.02 Values are given as mean ± SD (n=3) for each extract. Table 3. Peaks band characteristic on FTIR spectrum for H.Rosa Sinensis extracts. Group Frequencies (cm-1) Funtional group/assignment 1270 - 1230 Aromatic ethers, C-O stretch 1380 - 1350 Aliphatic nitro compounds/ nitrate ion 1555 - 1485 Aromatic nitro compounds 1650 - 1600 Quinone or conjugated ketone 1680 - 1630 Amide 1740 - 1725 Aldehyde 1725 - 1700 Carboxylic acid 1870 - 1820 Five-membered ring anhydride 3250 - 3650 Hydroxyl/ Amino group 3350 - 3320 Imino compounds, NH stretch 3490 - 3430 Heterocyclic amine, NH stretch 3670 - 3550 Non-hydrogen-bonded hydroxyl group Phytochemical Screening and Total Phenolics and Flavonoids Contents MedPharmRes, 2017, Vol. 1, No. 1 56 MedPharmRes, 2017, Vol. 1, No. 1 Truong et al. Figure 2. Calibration curve of gallic acid standard and rutin standard. Determination of total phenolics, gallic acid at concentrations of 20, 40, 60, 80, 100 µg·L-1 concentrations of 20, 40, 60, 80, 100 µg·L-1, y = 12,925x - 0,0523, R² = 0,989. Figure 3. Comparison of FTIR spectrum for four different H.Rosa Sinensis leaves extracts band positions. Figure 4. FTIR spectrum for H.Rosa Sinensis methanol leaves extract band positions 4. CONCLUSION is the screening step would help to identify new drug candidates. This study indicates that the extracts obtained from the leaves of H. Rosa Sinensis L. have the high value ascertains the potency of extracts from H. Rosa Sinensis as potential pharmaceutical sources. Therefore, the extract from H. Rosa Sinensis is worthy of further studies on chemotherapeutic activities and potential effects in vivo need to be further tested. 57 ACKNOWLEDGEMENT This study was supported by research funds of the Student project from International University Ho Chi Minh city-Vietnam National University (VNU-HCM) under grant number SV2017-BT-01. REFERENCE 1. Bhattacharya A, Vitaly Citovsky. The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection. Molecular Plant Pathology. 2010;11(5):705-719. 2. Charity Coury, Ann M. Dillner. 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