The Effect of Extraction Method on Total Phenolic Content and Antioxidant Activity of

Salam Leaves (Syzygium polyanthum) using DPPH (1,1-Diphenyl-2-Picrylhidrazil)

Extraction is an important step in separating bioactive compounds from the plant. The selection of
extraction technique is also important in the standardization of herbal products, for if not observed it can
remove the desired soluble constituents. One of Indonesia’s endemic plants which have been proven to
have an antioxidant activity is Salam leaves (Syzygium polyanthum). This study aims to determine the
effect of extraction method on total phenolic content and antioxidant activity of S. polyanthum leaves by
counteracting free radicals mechanism using DPPH. The S. polyanthum leaves were extracted by
maceration, soxletation, and infusa methods, and tested for the total phenolic content and the antioxidant
activity. The results showed that the total phenolic content of each method extration is different. The total
of the phenolic content extraction by maceration, soxhlet, and infusa method respectively were
338.62±21.3; 227.72±21.6; and 144.48±8.2 mgGAE/g. The best antioxidant activity was maceration
method with IC50 17.53±0.11 µg/mL followed by soxhlet and infusa which were 18.73±0.31 and
40.26±0.18 µg/mL. The research conclusion is that the extraction method has an effect on the total phenol
and antioxidant activity of S. polyanthum leaves.

Processing of citrus peel for the extraction of flavonoids for biotechnological applications

Flavonoids are extra nutritional constituents that naturally occur in small quantities in plants. They are a
family of polyphenolic compounds that are widespread in nature and are consumed as part of the human
diet in significant amounts. The diversity in their structure and bioactivity of flavonoids make these
compounds an interesting candidate for biotechnology based research. Extraction of flavonoids from
citrus employing the use of various techniques such as chemical and physical methods is attempted in this
write up. The biotechnological potential of flavonoids is not currently exploited to its maximum since
extraction procedures are in developing phase. The current knowledge on the sources of citrus flavonoids
and their potential activities in alleviating human health is also discussed.

Direct ultrasound-assisted extraction and characterization of phenolic compounds from fresh

houseleek (Sempervivum marmoreum L.) leaves

The effects of ultrasound power and frequency on the yield of total extractive substances (TES), total
phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity (AOA) of fresh houseleek
leaves extracts obtained by direct ultrasound-assisted extraction (DUAE) were studied. Preliminary
extraction of plant material was performed using methanol, acetone and 2-propanol by Soxhlet extraction.
It was found that maximum TES yield could be obtained by methanol extraction (2.91±0.02), followed by
acetone and 2-propanol with a TES yield of 2.32±0.01 and 2.01±0.03 g per 100 g of fresh plant material,
respectively. In the fresh houseleek leaves extracts obtained by DUAE and methanol as the chosen
solvent, TPC, TFC and AOA were in the ranges of: 40.5–85.9 mg gallic acid/g dry extract, 12.7–19.3 mg
rutin/g dry extract and 24.6–108.2μg/ml, respectively. The results showed that the increase in the
ultrasound power and extraction time have positive and significant (p < 0.05) effects on the TPC, TFC
and AOA, while the increase in the ultrasound frequency leads to a decrease in the TPC, TFC and AOA
of the extracts. A chromatographic analysis of crude extract identified the following: kaempferol 3-O-
(6’’-O-malonylglucoside)- 7-O-glucosyde, kaempferol 3-O-glucoside-7-O-rhamnoside, luteolin 5-O-(6’’-
O-malonylglucoside), kaempferol 3-O-(6’’-O-acetylglucoside)-7-O-rhamnoside, genkwanin 5-O-
glucoside, luteolin 5-O-(6’’-O-malonylglucoside), kaempferol 3-O-(6’’-O-malonylglucoside), kaempferol
3-O-rhamnoside, quercetin, genkwanin 4’-O-glucoside and hyperoside. [Project of the Serbian Ministry
of Education, Science and Technological Development, Grant no. 172047]

Antioxidant Activity and Phenolic Composition of Citrus Peel and Seed Extracts

A possible way to valorize citrus peels and seeds, which are byproducts of the juice extraction industry, is
to use them as natural antioxidants. The antioxidant activity of several citrus peel and
seed extracts obtained either by methanol extraction (free phenolic compounds) or by alkaline hydrolysis
(bound phenolic compounds) was tested in a model system based on accelerated citronellal oxidation.
Generally, seeds possessed greater antioxidant activity than peels. The composition of all tested samples
was studied by HPLC:  methanol extracts are rich in flavones and glycosylated flavanones, whereas
hydrolyzed extracts contain mainly phenolic acids and flavonols. The phenolic composition of
some citrus peels and seeds was described for the first time. No clear relationship could be shown
between the antioxidant activity and the phenolic composition of the extracts.

Phenols in Citrus Peel Byproducts. Concentrations of Hydroxycinnamates and

Polymethoxylated Flavones in Citrus Peel Molasses

In addition to the main flavanone glycosides (i.e., hesperidin and naringin) in citrus peel,
polymethoxylated flavones and numerous hydroxycinnamates also occur and are
major phenolic constituents of the molasses byproduct generated from fruit processing. Although a small
number of the hydroxycinnamates in citrus occur as amides, most occur as esters and are susceptible to
alkaline hydrolysis. This susceptibility to alkaline hydrolysis was used in measuring the concentrations of
hydroxycinnamates in citrus peel molasses. The highest concentrations of hydroxycinnamates occurred in
molasses of orange [C. sinensis (L.) Osbeck] and tangerine (C. reticulata Blanco.) compared to grapefruit
(C. paradisi Macf.) and lemon [C. limon (L.) Burm.]. Concentrations of two phenolic glucosides, phlorin
(phloroglucinol-β-O-glucoside) and coniferin (coniferyl alcohol-4-β-O-glucoside), were also measured.
Measurements of the polymethoxylated flavones in molasses from several tangerine and orange varieties
showed that these compounds occurred in the highest amounts in Dancy tangerine, whereas samples from
two other tangerine molasses contained significantly lower levels, similar to those in the molasses
samples from late- and early/mid-season oranges.
Converting citrus wastes into value-added products: Economic and environmently friendly
approaches

Citrus fruits, including oranges, grapefruits, lemons, limes, tangerines, and mandarins, are among the
most widely cultivated fruits around the globe. Its production is increasing every year due to rising
consumer demand. Citrus-processing industries generate huge amounts of wastes every year, and citrus
peel waste alone accounts for almost 50% of the wet fruit mass. Citrus waste is of immense economic
value as it contains an abundance of various flavonoids, carotenoids, dietary fiber, sugars, polyphenols,
essential oils, and ascorbic acid, as well as considerable amounts of some trace elements. Citrus waste
also contains high levels of sugars suitable for fermentation for bioethanol production. However,
compounds such as D-limonene must be removed for efficient bioethanol production. The aim of the
present article was to review the latest advances in various popular methods of extraction for obtaining
value-added products from citrus waste/byproducts and their potential utility as a source of various
functional compounds.

Extraction and quantification of polyphenols from kinnow (Citrus reticulate L.) peel using
ultrasound and maceration techniques

An investigation was carried out to extract polyphenols from the peel of kinnow (Citrus reticulate L.) by

maceration and ultrasound-assisted extraction (UAE) techniques. The antioxidant potential of these
polyphenols was evaluated using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-
picrylhydrazyl (DPPH), and superoxide radical scavenging assays; and their antimicrobial activity was
assessed against bacterial strains Staphyloccoccus aureus, Bacillus cereus, and Salmonella typhimurium.
The highest extraction yield was obtained through the solvent ethanol at 80% concentration level,
whereas UAE was a more efficient technique and yielded comparatively higher polyphenol contents than
maceration. Maximum polyphenols were extracted with 80% methanol [32.48 mg gallic acid equivalent
(GAE)/g extract] using UAE, whereas minimum phenolics (8.64 mg GAE/g extract) were obtained with
80% ethyl acetate through the maceration technique. Elevated antioxidant activity of kinnow peel extracts
was exhibited in three antioxidant assays, where 80% methanolic extracts showed the highest antioxidant
activity (27.67 ± 1.11mM/100 g for FRAP) and the highest scavenging activity, 72.83 ± 0.65% and
64.80 ± 0.91% for DPPH and superoxide anion radical assays, respectively. Strong correlations between
total polyphenols and antioxidant activity were recorded. Eleven phenolic compounds—including
five phenolic acids and six flavonoids—were identified and quantified by high performance liquid
chromatography. Ferulic acid and hesperidin were the most abundant compounds whereas caffeic
acid was the least abundant phenolic compound in kinnow peel extracts. Maximum inhibition zone was
recorded against S. aureus (16.00 ± 0.58 mm) whereas minimum inhibition zone was noted against S.
typhimurium (9.00 ± 1.16 mm). It was concluded that kinnow mandarin peels, being a potential source
of phenolic compounds with antioxidant and antimicrobial properties, may be used as an ingredient for
the preparation of functional foods.
Green solvents and Ultrasound-Assisted Extraction of bioactive orange (Citrus sinensis)
peel compounds

Byproducts such as orange peel have potential uses because of their bioactive compounds, which are
important for their potential to reduce the risk factors of diseases caused by aging. The lack of effective
techniques and the high levels of pollution produced by the conventional extraction of bioactive
compounds using organic solvents have highlighted the need to enhance the ‘green chemistry’ trend. This
study evaluates the use of ultrasound to extract bioactive compounds from orange peel. The antioxidant
capacity, phenolic content, ascorbic acid, total carotenoids, and HPLC profile of phenolic compounds
from orange peel extracts were obtained by a physicochemical evaluation. The results demonstrate that
the optimal conditions for the ultrasound-assisted extraction of bioactive orange peel compounds were a
power of 400 W, a time of 30 min, and 50% ethanol in water. These conditions were used to obtain a total
carotenoid concentration of 0.63 mg ß-carotene/100 g, vitamin C concentration of 53.78 mg AA/100 g,
phenolic concentration of 105.96 mg GAE/100 g, and antioxidant capacity of ORAC = 27.08 mM TE and
TEAC = 3.97 mM TE. The major phenolic compound identified in all orange peel extracts was
hesperidin, with a maximum concentration of 113.03 ± 0.08 mg/100 g.

Effect of ultrasound assisted extraction on the antioxidant activity and polyphenols from
lime (Citrus aurantifolia) peel

Lime peel is a rich source of polyphenols including phenolic acids and flavonoid.
The ultrasound assisted extraction method has been widely applied in extraction of
polyphenols from plant materials due to its ability to help in reducing extraction times and
increasing extraction yields. However, there have been a few reports on the effects of solvent
types and solvent concentrations on the polyphenols by the ultrasound assisted extraction.
The aim of this work was therefore to investigate the effects of solvent types (methanol,
ethanol and acetone and concentrations (20, 50 and 80% v/v) using ultrasound assisted
extraction on the antioxidant activity, total phenolic content, total flavonoid content and
flavanone glycosides (hesperidin and naringin) of lime peel. The results showed that the lime
peel extract obtained from using 80% acetone provided the highest antioxidant activity (IC50
by DPPH radical scavenging 4.32 mg/ml), total phenolic content (1,338.18 mg of gallic acid
equivalent/100 g DW), total flavonoid content (781.89 mg of quercetin equivalent/100 g DW)
and flavanone glycosides (622.53 mg of hesperidin and 53.39 mg of naringin/100 g DW).
Therefore, 80% acetone was regarded as a suitable solvent for extraction of polyphenols by
ultrasound assisted extraction from lime peel.
Extraction of phenolic compounds from lime peel waste using ultrasonic-assisted and
microwave-assisted extractions

Microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) have been studied to

extract natural phenolic compounds from lime peel wastes. A response surface methodology with a three-
level and three-factor Box-Behnken design was used to predict the optimal conditions of each extraction
parameter based on the total phenolic contents, antioxidant activities and the half maximal inhibitory
concentration. The results showed that the optimal predicted MAE was obtained at 55% ethanol
concentration, 140 W microwave power for 45 s with 8 repeats of the extraction step. Meanwhile, the
optimal predicted UAE was at 55% ethanol concentration, 38% amplitude and 4 min extraction time.
UAE was the more effective method to extract the total phenolics (54.4 mg GAE/g) with high antioxidant
activity and saving of time of 33% compared with MAE. Therefore, the natural phenolic extract from
lime peel waste might be available as a source of active and functional compounds for pharmaceutical
and food products.

Low Power Ultrasound-Assisted Extraction of Phenolic Compounds from Mandarin

(Citrus reticulata Blanco cv. Sainampueng) and Lime (Citrus aurantifolia) Peels and the
Antioxidant

Mandarin and lime peels are rich sources of phenolic compounds including phenolic acids and flavonoids.
The ultrasound-assisted extraction (UAE) proved to be an effective method in the extraction of phenolic
compounds from plants due to its ability to save extraction time and increase extraction yields. In this
research, factorial randomized complete experimental design has been used to investigate the effect of
various factors on UAE using low power (50.93 W). Influence of citrus peel types and solvent types and
concentrations on the total phenolic compounds, total flavonoids, and flavanone glycosides (hesperidin
and naringin) were studied. Antioxidant activity of the extracts was measured by ferric reducing
antioxidant potential (FRAP), azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and 1,1-
diphenyl-2-picrylhydrazyl (DPPH) assays. The optimum extraction conditions occurred when mandarin
peel and 80 % acetone were used. In those conditions, the extract contained 3,083.61 mg gallic acid
equivalent (eq) 100 g−1 dry weight (DW) of total phenolic compounds, 2,539.82 mg quercetin eq
100 g−1 DW of total flavonoid, 1,374.20 mg 100 g−1 DW of hesperidin, and the measured antioxidant
activity was 0.61 mg mL−1 of DPPH IC50, 1.12 mg mL−1 of ABTS IC50, and 2,521.47 mg trolox eq
100 g−1for FRAP. A strong correlation between total phenolic content and antioxidant activity was also
observed.