The Pharma Innovation Journal 2022; SP-11(3): 233-236

ISSN (E): 2277- 7695

ISSN (P): 2349-8242
NAAS Rating: 5.23 Extraction of clean-label food colourant from hibiscus
TPI 2022; SP-11(3): 233-236
© 2022 TPI
www.thepharmajournal.com
Received: 13-01-2022
Raghu A, Rita Narayanan, A Mangala Gowri, V Nithyalakshmi and
Accepted: 16-02-2022 Dorathy Pushparani
Raghu A
Abstract
College of Food and Dairy
Technology, Koduveli, Chennai,
As people become more conscious of the toxicity of synthetic colours, there is a greater demand for
India pigments derived from natural sources. Increase in consumer demand for natural products with no
chemical additives and certified dyes has necessitated the need to exploit food colourants of natural
Rita Narayanan origin. Anthocyanins are gaining popularity due to potential health advantages. Present investigations
Department of Food Processing were carried out to produce anthocyanin pigment as natural food colourant from Hibiscus rosa-sinensis.
Technology, College of Food and Solid-liquid extraction method was carried out with two different types of solvents viz. ethanol: distilled
Dairy Technology, Koduveli, water (1:1 v/v) and distilled water, acidified with food grade citric acid in the range from 0.5% to 3% in
Chennai, India order to recover maximum anthocyanin from the fresh flowers of Hibiscus rosa-sinensis. Acidified
distilled water extract with maximum TPC, TMA and AA value were chosen as a standardized extract
A Mangala Gowri and values were found to be 5180.50±0.02 mg GAE/100g, 191.73±0.01 mg C3G/100g, 87.92±0.06%.
Centralized Instrumentation This Natural food colourant finds applications in food products like dairy and beverage industry by
Laboratory, Madras Veterinary
replacing the existing synthetic colourants.
College, Vepery, Chennai, India

V Nithyalakshmi
Keywords: anthocyanin, natural colourant, H. rosa-sinensis, citric acid
Department of Food Process
Engineering, College of Food and 1. Introduction
Dairy Technology, Koduveli, Traditionally, several plants and their products have been used in foods as a mode of natural
Chennai, India preservative, flavoring agent as well as a remedy to treat some of the common ailments in
Dorathy Pushparani
human (Voon et al., 2012) [12]. Plants are rich source of bioactive compounds which were
Department of Food Chemistry responsible for the prevention and treatment of chronic health conditions such as hypertension,
and Food Processing, Loyola cardiovascular diseases, inflammation and cancer. Extracts of these bioactive components can
College, Chennai, India also be used as natural colorants for food because they are believed to be safe, and non-toxic to
human (Boo et al., 2012) [4] Among the bioactive components, Anthocyanins were identified
as a potential one responsible for beneficial properties such as antioxidant and antimicrobial
activities (Pires et al., 2019) [10]. The synthetic colouring additives in the food industry can be
replaced by anthocyanins which correspond to reddish–purple colour and present in most of
the flowers and fruits (Jabeur et al. 2017) [7].
Hibiscus rosa-sinensis from the family Malvaceae is abundantly flowering, perennial, woody
ornamental shrub widely found in the tropical regions. Studies on H. rosa-sinensis have
revealed to possess high amount of bioactive components and is recommended as an herbal
alternative to cure many diseases (Obi et al., 1998) [5]. Major anthocyanins present in old
strains of Hibiscus rosa sinensis L. is found to be cyaniding-3-ꞵ-sophoroside (Nakamura et al,
1990) [9] and the dark red flowers of H. rosa-sinensis are important source of cyanidin-3-
glucoside. In Ayurveda and ancient literatures, H. rosa-sinensis has been used in various
conditions like hypertension, pyrexia, liver disorders, antifertility activity, epilepsy, leprosy,
bronchial catarrh and diabetes.
The present study was focused on the extraction of crude anthocyanins from Hibiscus rosa
sinensis using two different solvents ie. distilled water and Ethanol: distilled water (50:50)
with 0.5 to 3% acidifying agent and comparing the extraction by analyzing total polyphenols,
total monomeric anthocyanin and antioxidant values. These results will demonstrate the
antioxidant activity and quantification of bioactive components present in hibiscus flower after
extraction and will be helpful for small-scale food business operators by reducing the usage of
artificial colouring agents thereby a new marketability trend will get created. these extracts can
Corresponding Author
be used as a potential natural food colourant in Food, Dairy and Beverage industries.
Raghu A
College of Food and Dairy
Technology, Koduveli, Chennai,
India
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2. Materials and Methods and 700nm against distilled water as blank solution using UV-
2.1 Materials Visible spectrophotometer (UV Mini – 1240).
2.1.1 Plant materials
Fresh Hibiscus rosa-sinensis flowers were obtained from the Anthocyanin pigment (cyanidin-3-glucoside equivalents,
local market, Chennai. The fresh flowers were examined for 𝐴𝐴 ×𝑀𝑀𝑀𝑀 × 𝐷𝐷𝐷𝐷 ×10^3
mg/L) =
Ɛ × 𝑙𝑙
quality and bright red flowers without any damage were
washed with distilled water and shade dried just to reduce
Where,
surface water content and stored in nylon bags at 4°C until
A = (A520nm – A700nm) pH 1.0 - (A520nm – A700nm) pH 4.5
further analysis.
MW (molecular weight) = 449.2 g/mol for cyanidin-3-
glucoside (cyd-3-glu)
2.1.2 Chemicals
DF = dilution factor
Double distilled water, Ethanol (AR 99%), Food grade citric
l = pathlength in cm
acid, Sodium carbonate, Folin-Ciocalteu reagent (FCR),
Ɛ = 26 900 molar extinction coefficient, in L × mol–1 × cm–1,
Potassium chloride, Sodium acetate, DPPH - 2, 2-diphenyl-1-
for cyd-3-glu.
picrylhydrazyl (Hi media), The reagents required for analysis
10^3 = factor for conversion from g to mg
were freshly prepared by adopting standard procedures.
2.2.5 Determination of Antioxidant activity (AA)
2.1.3 Solvent preparation
The free radical scavenging activity of the flower extract was
Solvent 1: Ethanol-50% is prepared by adding 500ml of
determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
distilled water with 500ml of Ethanol (AR 99%) in a standard
scavenging method modified from Alhakmani et al. (2013) [3].
measuring flask. After preparation of solvent 1 acidification is
Briefly, the mixture contained 1 mL of 0.1mmol DPPH
done by adding various amounts of Food grade citric acid
radical solution prepared in methanol and 0.1 mL of extract
from 0.5% to 3%.
solution is mixed with DPPH solution. The solution was
Solvent 2: Distilled Water Acidified with Food grade citric
rapidly mixed and incubated in dark at 37 °C for 20 min. The
acid from 0.5% to 3%
absorbance of each solution was measured at 517 nm against
0.1mmol DPPH solution in methanol as blank using UV/Vis
2.2 Methods
spectrophotometer.
2.2.1 Determination of pH
The pH of extracted samples was measured by using a bench- 𝐴𝐴𝐴𝐴−𝐴𝐴𝐴𝐴
top pH meter (Model Susima MP1 Plus). % Free radical scavenging activity = × 100
𝐴𝐴𝐴𝐴

2.2.2 Determination of Total soluble solids (TAA) Where, Ac – Absorbance of Control sample, As – Absorbance
Total soluble solids (TSS) (ºBrix) of extracted samples were of sample.
determined using a hand refractometer according to AOAC
(2000) at room temperature (25±1 ºC) expressed as ºBrix (0 - 2.2.6 Statistical analysis
90). VETSTAT software was used for statistical analysis and the
results were given as mean± standard deviation (SD).
2.2.3 Determination of total polyphenol content (TPC)
Total polyphenols present in the exaction were determined by 3. Results and Discussion
Folin-Ciocalteu reagent, following Bergmeier et al. (2014) [3]. 3.1 Effect of solvents on the Physico chemical properties of
Extract aliquots (50-100 μl) were transferred into the test flower of Hibiscus rosa-sinensis extract
tubes and the volumes were completed to 5 mL with distilled Physicochemical properties such as pH and TSS of hibiscus
water. After adding 0.20 ml Folin-Ciocalteu reagent and 0.5 extracts were determined and the results are given in table 1.
ml saturated aqueous sodium carbonate solution the tubes It is observed that the pH gradually decreased for hibiscus
were vortexed and absorbance of the mixtures was recorded extract. This can be attributed to the increase in the citric acid
after 20 min., at 765 nm, by using a UV-Visible concentration in solvent extraction method. Similar results
spectrophotometer (UV Mini – 1240). The amount of total were obtained by Arab et al. (2011) [1] who reported that pH
polyphenols was calculated as gallic acid equivalents from the of hibiscus extract increased with increased concentration of
calibration curve with gallic acid standard solution. Results citric acid.
were expressed as mg of total phenolic content (gallic acid The TSS of the hibiscus extract increased gradually with
equivalent) per gram of fresh flower (mg GAE 100 g-1dry increase in citric acid concentration. The acidified solvents
flower) degraded the cell membrane and dissolved the soluble solids
into the extract thereby, increasing the TSS content. The
2.2.4 Determination of total monomeric anthocyanin results in the present study ranged from 3.10 to 5.43 and are
(TMA) in concurrence to the study reported by Arab et al. (2011) [1]
Total monomeric anthocyanin content of the extract was who reported that TSS for Hibiscus roselle plant extract with
determined by pH differential method following AOAC 2% citric acid solution was 12 °Brix and 5 °Brix in aqueous
Official Method 2005.02. pH 1.0 buffer and pH 4.5 buffer solvent.
were made from potassium chloride (0.025M) and sodium
acetate (0.4M). 1 part of the test solution is mixed with 4 parts 3.2 Effect of solvents on the extraction of TPC and TMA
of each buffer solution in a 50ml volumetric flask (total and AA from the flowers of Hibiscus rosa-sinensis
diluted solution should be <10ml) and the absorbance was Extraction efficacy of ethanol with acidified water containing
measured within 20-50mins of preparation. The diluted test citric acid and distilled containing varying concentration of
portions at pH 1.0 and pH 4.5 both were measured at 520nm citric acid were compared for TPC and TMA and AA in the

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crude extract of hibiscus extract. As shown in table 2, TPC were not too acidic to cause partial hydrolysis of the acyl
and AA of hibiscus using ethanol: water acidified solvent had moieties in acylated anthocyanins as suggested by Andersen
higher affinity of extraction than acidified water extraction. and Markham, (2006). Hence the TPC and TMA content
However, it is noticed that the TMA in acidified water extract increased with increase in citric acid concentration which is
was significantly higher in aqueous acidified extract when concordant with the observations of Chandrasekhar et al.
compared to ethanol: water acidified extract. This result (2012) [6].
coincided with the findings of Vankar et al. (2010) [11] who However, at 3% citric acid concentration, both solvents
also observed that TMA of acidified aqueous extract was showed a decrease in extraction yield of TPC and TMA
higher than that of ethanolic extracts. content. This decrease might be due to the hydrolysis of the
The TPC and TMA of hibiscus crude extract had highly acyl moieties in acylated anthocyanins at higher concentration
significant yield due to the acidified solvents which degraded of acid in solvent medium (Arab et al., 2011) [1].
the cell membrane and dissolved phenolic components and The AA of the extract range was significantly higher in
stabilized them simultaneously. This may due to the aqueous acidified solvent which is concomitant to the work of
formation of flaviyum cationic salts in lower acidic medium Kruawan et al. (2006) [8] who also observed that DPPH
which serves as an ideal medium to extract and stabilize most radical scavenging effect of Hibiscus with hot water extract
of the TPC and TMA content in the extracts. But contents was about 93.12%.

Table 1: Treatment combination for Acidified solvent extraction

Treatments Ethanol (V/V) Distilled Water (V/V) Citric Acid (Food Grade)
T1 50% 50% 0.5%
T2 50% 50% 1%
T3 50% 50% 1.5%
T4 50% 50% 2%
T5 50% 50% 2.5%
T6 50% 50% 3%
T7 Nil 100% 0.5%
T8 Nil 100% 1%
T9 Nil 100% 1.5%
T10 Nil 100% 2%
T11 Nil 100% 2.5%
T12 Nil 100% 3%

Table 2: Effects of solvents on physico-chemical properties of Hibiscus rosa-sinensis extract

Treatments pH TSS(°Brix)
T1 2.43f±0.00 3.26a±0.02
T2 2.34e±0.00 3.54b±0.04
T3 2.22d±0.00 3.99c±0.04
T4 2.10c±0.00 4.42d±0.03
T5 1.97b±0.00 5.13f±0.04
T6 1.86a±0.00 5.43g ±0.02
T7 2.41f±0.01 3.10a±0.06
T8 2.33e±0.00 3.38b±0.05
T9 2.23d±0.00 3.83c±0.03
T10 2.11c±0.00 4.27d±0.03
T11 1.99b±0.00 4.73e±0.03
T12 1.88a±0.00 5.23f±0.08
F-value 1579.16** 333.04**
@Average of six trials
** statistically highly significant (P<0.01)
Means bearing various superscripts in the same column differs highly significantly (P<0.01)

Table 3: Effect of solvents on extraction of TPC, TMA, AA from the flowers of Hibiscus rosa-sinensis
Total Monomeric Anthocyanin Antioxidant Activity
Treatments Total Polyphenol content(mg GAE/100g)
(mgC3G/100g) (%)
T1 4996.36b ±0.01 159.17a±0.16 83.33d±0.03
T2 5069.78d±0.02 167.70d±0.02 85.44e±0.02
T3 5102.26f±0.02 172.04e±0.02 89.57h±0.02
T4 5153.37h±0.01 188.82g±0.01 92.42k±0.01
T5 5254.74l±0.01 190.49j±0.01 91.80j±0.01
T6 5185.78k±0.02 189.27h±0.01 90.15i±0.02
T7 4986.25a±0.03 160.49b±0.02 73.58a±0.05
T8 5004.36c±0.01 165.67c±0.01 78.76b±0.06
T9 5074.78e±0.02 173.27f±0.01 82.40c±0.03
T10 5122.26g±0.02 188.58g±0.01 85.76f±0.03
T11 5180.50j±0.02 191.73k±0.01 87.92g±0.06
T12 5176.46i±0.02 189.77i±0.15 85.56e±0.03
F-value 19886975.17** 38280.25** 24885.12**
@Average of six trials
** statistically highly significant (P<0.01)
Means bearing various superscripts in the same column differs highly significantly (P<0.01)

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Fig 1: Flow diagram for Extraction of crude anthocyanin recovery

4. Conclusion Technology. 2014;36(3):545-551.

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