Comparative Antibacterial Efficacy of Gomphrena Globosa L. Flower Using Hot Continuous and Sonication Extraction Methods

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INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 10, OCTOBER 2019 ISSN 2277-8616

Comparative Antibacterial Efficacy Of Gomphrena Globosa l. Flower Using Hot Continuous And Sonication Extraction Methods

Jesse Joel T, Levin Anbu Gomez, Priti Lata Murmu

Abstract: Gompherna globosa L. is a good source of betacyanin, and it is highly found in the pigment of the plant, even after drying, it retain its color for a longer period time. Betacyanins include the reddish to violet betalain pigments. Betacyanin were extracted from Gomphrena globosa l. flower using two methods of extraction. One is ultrasonic bath method, which use ethanol as solvent, which uses high frequency pressure(sound) wave to agitate the liquid. On the other hand, soxhlet uses methanol as a solvent, which is heated to reflux. Both the operations were compared to check the efficiency and yield of the product. Ultrasonic bath was more efficient than soxhlet, but soxhlet have higher yield than ultrasonic bath. After the product yield, HPLC is done for identification, quantification and molecular weight. Escherichia coli was used to test for antibacterial efficacy. Cefoxitin antibiotic disc was used to know the bacterial susceptibility. The extraction methods concentration was taken same (200, 250, 300, 350) µg/µl. The zone formation in the medium indicates that Gomphrena globosa resist the growth of bacteria, but when the Zone of inhibition was compared, the ultrasonic bath extract showed was more inhibition than Soxhlet extract.

Index Terms: Gomphrena globosa l. flower, frequency pressure wave, reflux, Antibacterial efficiency ——————————  ——————————

1. INTRODUCTION led to the production of many cultivars of globe amaranth such Motivation and BackgroundGomphrena globosa, commonly as those varying in flower colors that include purple, lavender, known as globe amaranth, makhmali, and vadamalli, is an magenta, red, orange, and white [1]. Carotenoids (specifically edible plant from the family Amaranthaceae. The round- on lycopene), anthocyanins and betacyanins are natural red shaped flower inflorescences are a visually dominant feature pigments found on fruits and vegetables. They pose and cultivars have been propagated to exhibit shades of antioxidant properties beneficial in the prevention of cancer magenta, purple, red, orange, white, pink, and lilac. Within the and cardiovascular disease. Recently natural coloring agents flower heads, the true flowers are small and inconspicuous. have attached increasing attention from the food industries as The flowers of globe amaranth have been used as a type of a substitute to the artificial coloring components which has herbal medicine, implying that they may contain essential oils. been discovered to cause negative impact to humans health Based on the hypothesis that globe amaranth flowers produce upon consumption there is a growing interest in the natural red and emit volatiles, the first objective of this study was to pigments in food science, due to chronic disease preventive determine what volatile compounds are emitted from the effects stability of the natural pigments should be concerned flowers of globe amaranth [1]. as it might affect the saturated of the color [2] The plant Globe amaranth is a horticultural species native to Central Gomphrena globosa grows well in different parts of America. The flower heads are tiny it features globe-shaped, Bangladesh and used traditionally for many medicinal clover-like flower heads, and 4 cm long at maturity. As a purposes. To verify the traditional uses the crude methanol tropical annual plant, globe amaranth continually blooms extract and its hexane, carbon tetrachloride, chloroform and throughout summer and early fall. Thus, it has been a favorite aqueous soluble fractions were subjected to antimicrobial, landscape plant in annual beds, rock gardens, and garden antioxidant and brine shrimp lethality bioassays. Among all borders. After being removed from the plant, the papery- extractives the hexane soluble fraction exhibited the highest textured flowers of globe amaranth can retain their color and antioxidant activity having IC50 value 13.17±0.308 µg/ml and shape for a long time. G. globosa is an outcrossing species the crude methanol extract showed significant antioxidant that is pollinated by butterflies, bees, and other insects. Floral activity with IC50 value 20.35±0.360 µg/ml [3]. G. globosa is volatiles likely play a significant role in the reproductive native to Central America including regions of Brazil, Panama, success of the plant by promoting the attraction of pollinators and Guatemala, but is now grown globally [4]. As a tropical the globe amaranth has also been popular as a cut flower and annual plant, G. globosa blooms continuously throughout a dried. Continuous germplasm selection and breeding have summer and early fall. It is very heat tolerant and fairly drought resistant, but grows best in full sun and regular moisture. ———————————————— Despite the fact that globe amaranth is an important annual  Dr. Jesse Joel T, Corresponding Author, Assistant Professor, Department of Biotechnology, Karunya Institute of Technology flower plant and its flowers have been the subject of many and Sciences, Coimbatore – 641114. Tamil Nadu. Mob: biological studies, little, if anything, is known about its floral 9487733707, E-Mail: [email protected]. volatile chemistry. Globe amaranth is an outcrossing species.  Dr. Levin Anbu Gomez, Co-Author, Assistant Professor, Flowers of globe amaranth are visited by butterflies, bees, and Department of Biotechnology, Karunya Institute of Technology other insects. For plants that are cross-pollinated, floral and Sciences, Coimbatore – 641114. Tamil Nadu. Mob: volatiles often play an important role in their reproductive 9487733707, E-Mail: [email protected]  Ms. Priti Lata Murmu, Co-Author, is currently pursuing success by functioning as a cue to attract pollinators. In Master’s degree program in Biotechnology in Karunya addition, the flowers of globe amaranth have been used as a Institute of Technology and Sciences, India. (This information type of herbal medicine, implying that they may contain is optional; change it according to your need.) essential oils. Based on the hypothesis that globe amaranth flowers produce and emit volatiles, the first objective of this study was to determine what volatile compounds are emitted 2311 IJSTR©2019 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 10, OCTOBER 2019 ISSN 2277-8616

from the flowers of globe amaranth [1]. In Hawaii, it is calcium dipicolinate in bacterial spores, including Bacillus commonly used in long-lasting leis since it retains its shape anthracis and B cereus [10]. Betanin is commercially used as and color after drying. In Nepal, the flower is used to make a a natural food dye. It can cause beeturia (red urine) and red garland during Bhai Tika, last day of Tihar. The garland is put feces in some people who are unable to break it down. The around the brother's neck by their sister for protection. This interest of the food industry in betalains has grown since they plant is popular in landscape design and cutting gardens for its were identified by in-vitro methods as antioxidants, which may vivid colors and color retention. The edible plant G. globosa protect against oxidation of low-density lipoproteins [11]. The has been used in herbal medicine. In Trinidad, the flowers are linear and nonlinear optical studies of a natural dye betacyanin boiled to make a tea which is used for baby gripe, oliguria, extracted from red beet root in the presence of silver nano cough and diabetes. Caribbean folk medicine historically used particles in colloidal solution. We synthesized silver nano globe amaranth to relieve prostate and reproductive problems particles and characterized by XRD and HRTEM. We show [5]. The purple inflorescences have been used as a remedy for how appropriate concentration of silver nanoparticles can several respiratory inflammation conditions including bronchial enable tuning of dye fluorescence efficiency. Nonlinear asthma, acute and chronic bronchitis, and whooping cough [6]. properties are studied using open aperture Z scan technique Recommended uses in traditional medicine range from the with Nd: YAG laser (532 nm, 7 ns, 10 Hz). We show treatment of respiratory diseases, jaundice, urinary system modification of nonlinear properties for the dye to the desired conditions, and kidney problems [4]. The flowers of G. globosa level can be achieved in the presence of silver nanoparticles. are rich in betacyanins which have a wide range of High nonlinearity we also demonstrated in PVA/Ag applications as additives and supplements in the food industry, nano/Betacaine composite films. Theoretical analysis is cosmetics, and livestock feed. Stable between pH 3 and 7, the performed using model based on nonlinear absorption of betacyanins in globe amaranth are well suited to be used as materials and scattering of metal nanoparticles [12]. The natural food dye and have a red-violet color. Betacyanins have flowers of globe amaranth have been used as a type of herbal displayed interesting medicinal characteristics as powerful medicine, implying that they may contain essential oils. Based antioxidant and chemo preventive compounds in both in vitro on the hypothesis that globe amaranth flowers produce and and in vivo models. G. globosa inflorescences extract emit volatiles, the first objective of this study was to determine decreased nitric oxide content in cultured macrophages [7] At what volatile compounds are emitted from the flowers of globe least twenty-seven phytochemicals have been detected in G. amaranth [1] globosa including six phenolic acid derivatives and fifteen specific flavonoids. The most abundant phenolic compounds 2 MATERIALS AND METHODS present are flavonoids. A major phenol was found to be kaempferol 3-O-rutinoside based on chromatographic and 2.1 Sample collection mass spectrometry techniques. Gomphrenol derivatives also Gomphrena Globosa L. Flowers was obtained from contribute to phenolic content. Other flavonols include Paravattani, Trissur in Kerala. Its authentication was done quercetin, kaempferol, and isorhamnetin derivatives. [6] in Tamil Nadu Agriculture University (TNAU), Coimbatore. Betacyanin identified in globle amaranth are gomphrena, isogomphrenin II, and isogomphrena III [4]. Betalains are a 2.2 Preparation of Extracts class of red and yellow indole-derived pigments found in plants The flower was washed with tap water and was dried in the of the Caryophyllales, where they replace anthocyanin hotair oven for 2 to 3 days. Flower was made into powder by pigments. Betalains also occur in some higher order fungi. grinding it in kitchen blender. The flower was grinded until a They are most often noticeable in the petals of flowers, but fine powder was obtained. The fine powder was stored in dried may color the fruits, leaves, stems, and roots of plants that and dark place at room temperature for further process. Two contain them. They include pigments such as those found in extraction methods were carries out: Soxhlet extraction beets. The name ―betalain‖ comes from the Latin name of the methodThe method used for extraction was soxhlet, it was common beet (beta vulgaris) from which betalain was first described in [13]. With some modification. The solvent used extracted. The deep red color of beets, bougainvillea, for extraction was methanol, it was described in [14]. Flower amaranth, +and many cacti results from the presence of powder was taken 20 g and it was packed with (Whatmann betalain pigments. The particular shades of red to purple are No.1filter paper) and the packet was placed within the thimble distinctive and unlike that of anthocyanin pigments founds in above the round-bottom distillation flask (500 ml). Methanol most plants. They are most often noticeable in the petals of (300 ml) was added and the solvent was boiled at 60ºC under flowers, but may color the fruits, leaves, stems, and roots of reflux for 8 hours (Fig. 2). After the extraction the solvent and plants that contain them. They include pigments such as those extract were separated by using rotary vacuum evaporator found in beets [8]. The most heavily studied betalain is (Fig. 3). The remaining extract was poured in the petri dish betanin, also called beetroot red after the fact that it may be and keep in the hot air oven (64˚C) to evaporate the excess of extracted from red beet roots. Betanin is a glycoside, and methanol (Fig. 5). After complete sample dryness, the hydrolyzes into the sugar glucose and betanin. It is used as a achieved solid residue was removed from the oven and food coloring agent, and the color is sensitive to pH. Other allowed to cool before weighting. The extraction yield was betalain known to occur in beets are isobetanin, probetanin, noted and stored in the refrigeration until further and neobetanin. The color and antioxidant capacity of betanin analysisUltrasonic bath extraction methodThe method used for and indicaxanthin (betaxanthin derived of L-proline) are extraction was ultrasonic bath, it was described in [15] with affected by dielectric microwave heating [9]. Addition of TFE some modification. The flower powder was taken in a beaker (2, 2, 2- trifluoroethanol) is reported to improve the hydrolytic (500 ml), ethanol and water (150:150,v:v) were used as stability of some betalains in aqueous solution. Furthermore, solvent for extraction, the beaker was kept in the ultrasonic a betanin-europium(III) complex has been used to detect water bath which uses high frequency pressure(sound) wave

to agitate the liquid (Fig. 4). After ultrasound it was filtered with (Whatmann No.1filter paper) discarded the solid and only the supernatant was taken. The ﬁltered solution was dried in an oven, ﬁrstly at 60°C to evaporate the ethanol, then at 100°C to evaporate the residual water (Fig. 6). After complete sample dryness, the achieved solid residue was removed from the oven and allowed to cool before weighting. The extraction yield was noted .and was stored in the refrigeration until Fig. 3 further analysis (Fig. 7).

2.3 HPLC: Chromatographic analyses were carried out on a Luna C18 column (250 ×4.6 mm, 5 mm particle size; Phenomenex, Macclesfield, UK). The mobile phase consisted of two solvents: water/acetic acid (1%) (A) And methanol (B), the flow rate was 1 mL/min and the injection volume 40mL. Spectral data from all peaks were accumulated in the range 240–650 nm, and chromatograms were recorded at 280, 340, 470 and 540 nm [16]. The column used for HPLC was c18 and the sample which was given for analysis was flower extract, sample was diluted 10mg in 1ml and was given for HPLC analysis. Fig. 4

2.4 Culture preparation About 0.65 g of nutrient broth was taken and was suspended in 50ml of distilled water in a conical flask. Then it was sterilized by autoclaving at 15 lbs pressure 121°C for 15 minutes. And after nutrient broth is cooled, Escherichia Coli (gram-negative) was inoculated into the broth. And was keep in incubator at 36°C overnight for the bacteria to grow.

2.5 Antibacterial activity Well diffusion method was performed for bacterial activity. Escherichia Coli (gram-negative) was used as test Fig. 5 organism, which was grown overnight. Cefoxitin antibiotic disc was used to know the bacterial suppressibility. The zone of inhibition at various concentration was measured and noted.

2.6 Figures

Fig. 6

Fig. 1

Fig. 2

Fig. 7

3 RESULTS the component which was eluted first was high polar and the Recovery of extraction Gomphrena globosa extraction other component are less polar. At 254 nm wavelength it done by two method soxhlet and ultrasonic bath. In soxhlet shows eight components are high polar and the first three are method, 20g of flower power was taken and 500ml of more comparing other and the other which is eluted late are methanol was poured in round-bottom distillation flask it less polar. The peak indicates separation of individual used as a solvent for extraction. Methanol was boiled at component. The highest and sharpest peak it indicates the 60ºC under reflux for 8 hours. And in ultrasonic bath very well separation of component. The bridge like peak method, 20g of flower power was taken, ethanol and water indicate there is no proper separation of component. (150:150; v:v) was added into the beaker, it was kept in the Comparing to soxhlet extraction, the ultrasonic bath extraction water bath which uses high frequency pressure(sound) has proper separation at 254 nm (Fig. 9). wave to agitate the liquid for 40 minutes. The higher yield extraction of flower component was obtained by refluxing Antibacterial activity with ethanol. The extraction was done for 40 minutes and Antibacterial test was done using both the extraction to see its yield was 1.71g. In ultrasonic bath, used high frequency which has better zone of inhibition (Fig. 10A, 10B) and the pressure (sound) wave to agitate the methanol for 40 bacteria used was Escherichia coli. minutes and its yield was 1.5g. Therefore, soxhlet extraction yield was found more comparing to ultrasound bath extraction.

HPLC analysis: The column used for HPLC was c18 and the sample which was given for analysis was flower extract, the sample was diluted 10mg in 1ml it was done for both the sample and was given for HPLC analysis.

Fig. 10A

Fig. 8 Here we observe that at 218 nm wavelength it shows that the two component which was eluted first had higher polarity and other component which took more time to eluted have less Fig. 10B polarity (Fig. 8). The zone formation in the medium indicates that Gomphrena globosa resist the growth of bacteria, both the extraction methods concentration was taken same (200, 250, 300, 350) µg/µl to check the zone of inhibition, but when it was compared ultrasonic bath extraction method, zone of inhibition was more than the Soxhlet (Table. 1).

Zone of inhibition (mm) S. Extraction Concentration (µg/µl) No methods Control 200 250 300 350 (Cefoxitin)

1 Ultrasonic bath 23 5 9 12 15 Fig. 9 At 254 nm wavelength it shows three components was eluted 2 Soxhlet 23 0 3 6 8 first which have high polarity and the other component have less polarity. From figure 11, at 218 nm wavelength it shows Table. 1

4 DISCUSSION C. Ling, ―Natural red pigments from plants and their In this study, Gomphrena globosa L. flower showed health benefits: A review,‖ Food Rev. Int., vol. 34, no. 5, antibacterial efficacy on Escherichia coli. Gomphrena globosa pp. 463–482, 2018. flower component was extracted by ultrasonic bath which [3] M. Hamiduzzaman and A. Z. Azam, ―Antimicrobial, showed antibacterial activity [7], [17]. The component which Antioxidant and Cytotoxic Activities of Gomphrena act as an antibacterial is betacyanin. Gomphrena globosa globosa (L.),‖ Bangladesh Pharm. J., vol. 15, no. 2, pp. compound are widely used in many places like in dye 183–185, 2012. industries and food chemistry. Chromatographic analyses [4] C. L. Roriz, L. Barros, A. M. Carvalho, C. Santos- were carried out on a Luna C18 column (250 4.6 mm, 5 mm Buelga, and I. C. F. R. Ferreira, ―Pterospartum particle size; Phenomenex, Macclesfield, UK). The mobile tridentatum, Gomphrena globosa and Cymbopogon phase consisted of two solvents: water/acetic acid (1%) (A) citratus: A phytochemical study focused on antioxidant and methanol (B), starting with 20% B and using a gradient to compounds,‖ Food Res. Int., vol. 62, pp. 684–693, obtain 50% at 30 min, 90% at 40 min, 95% at 45 min. The flow 2014. rate was 1 mL/min and the injection volume 40 ml. Spectral [5] C. Lans, ―Ethnomedicines used in Trinidad and Tobago data from all peaks were accumulated in the range 240–650 for reproductive problems,‖ J. Ethnobiol. Ethnomed., nm, and chromatograms were recorded at 280, 340, 470 and vol. 3, pp. 1–12, 2007. 540 nm. This was the procedure which was used for [6] L. R. Silva et al., ―Phytochemical investigations and separation and structural characterization of betacyanin [16]. biological potential screening with cellular and non- The separation of component from gomphrena globosa l. cellular models of globe amaranth (Gomphrena flower, the column which was used was c18 reversed phase globosaL.) inflorescences,‖ Food Chem., vol. 135, no. column for the separation of component. The extraction of 2, pp. 756–763, 2012. flower, leaves, stem etc. was done by Soxhlet and it is one of [7] C. L. Roriz, L. Barros, M. A. Prieto, P. Morales, and I. the best methods for extraction [13]. Therefore, the soxhlation C. F. R. Ferreira, ―Floral parts of Gomphrena globosa was done for the extraction of component from Gomphrena L. as a novel alternative source of betacyanins: globosa. Antibacterial test was done for both the methods, Optimization of the extraction using response surface concentration was taken same (200, 250, 300, 350) µg/µl to methodology,‖ Food Chem., vol. 229, pp. 223–234, check the zone of inhibition, but when it was compared with 2017. Soxhlet extraction method the zone of inhibition was found [8] D. Strack and V. Wray, ―Recent Advances in Betalain more in ultrasonic bath. Analysis,‖ Caryophyllales, vol. 62, pp. 263–277, 2011. [9] L. C. P. Gonçalves, B. M. Di Genova, F. A. Dörr, E. 5 CONCLUSIONS Pinto, and E. L. Bastos, ―Effect of dielectric microwave In this study, Gomphrena globosa L. Flower was extracted by heating on the color and antiradical capacity of two different method, one was ultrasonic bath which used betanin,‖ J. Food Eng., vol. 118, no. 1, pp. 49–55, ethanol and water (150:150; v: v) and on the other hand 2013. soxhlet which used methanol as a solvent. Both the method [10] L. C. P. Gonçalves, S. M. Da Silva, P. C. DeRose, R. A. had different working soxhlet reflux the methanol for extraction Ando, and E. L. Bastos, ―Beetroot-Pigment-Derived and ultrasonic bath used ethanol and water as solvent which Colorimetric Sensor for Detection of Calcium use high frequency pressure (sound) for agitating of liquid. The Dipicolinate in Bacterial Spores,‖ PLoS One, vol. 8, no. yield was more in Soxhlet as it heated the ethanol to extract all 9, pp. 1–6, 2013. the component from flower sample. HPLC analysis was done [11] L. Tesoriere, M. Allegra, D. Butera, and M. A. Livrea, for both the method, but ultrasonic bath had more clear ―Absorption, excretion, and distribution of dietary separation of component compared to Soxhlet. Antibacterial antioxidant betalains in LDLs: Potential health effects test was done, both methods concentrations were same but of betalains in humans,‖ Am. J. Clin. Nutr., vol. 80, no. zone of inhibition was more in ultrasonic bath than Soxhlet. 4, pp. 941–945, 2004. So, it was concluded that ultrasonic bath was more efficiency [12] A. Sarkar, A. Thankappan, and V. P. N. Nampoori, than Soxhlet. It highlights that the use of Gomphrena globosa ―Effect of silver nanoparticles on fluorescence and as a source of betacyanin compounds in an optimized nonlinear properties of naturally occurring betacyanin ultrasonic bath assist in the production of extracts with dye,‖ Opt. Mater. (Amst)., vol. 39, pp. 211–217, 2015. important colorant properties, thus with high potential to be [13] M. A. Ali, T. A. Al-hattab, and I. A. Al-hydary, ―E used as natural colorant additives. Xtraction O F D Ate P Alm S Eed O Il ( P Hoenix D Actylifera ) B Y S Oxhlet a Pparatus,‖ Int. J. Adv. Eng. Technol., vol. 8, no. 3, pp. 261–271, 2015. ACKNOWLEDGMENT [14] L. Paniwnyk, E. Beaufoy, J. P. Lorimer, and T. J. 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