Gallic Acid: Review of the Methods of Determination and Quantification

Felipe Hugo Alencar Fernandes & Hérida Regina Nunes Salgado

To cite this article: Felipe Hugo Alencar Fernandes & Hérida Regina Nunes Salgado (2015): Gallic Acid: Review of the Methods of Determination and Quantification, Critical Reviews in Analytical Chemistry, DOI: 10.1080/10408347.2015.1095064
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Felipe Hugo Alencar Fernandes and Hérida Regina Nunes Salgado

School of Pharmaceutical Sciences, São Paulo State University, Araraquara, SP, Brazil

Corresponding Author Email: [email protected]


Gallic acid (3,4,5 trihydroxybenzoic acid) is a secondary metabolite present in most plants. This metabolite is known to exhibit a range of bioactivities including antioxidant, antimicrobial, anti- inflammatory and anticancer. There are various methods to analyze gallic acid including spectometry, chromatography, capillary electrophoresis, among others. They have been developed to identify and quantify this active ingredient in most biological matrices. The aim of this paper is to review the available information on analytical methods to gallic acid, as well as presenting the advantages and limitations of each technique.


Gallic acid, polyphenol, analytical methods.

1. Introduction

Considered one of the major phenolic acid, gallic acid (or gallate) is a benzoic acid of great importance for the formation of a so-called galatotanins-hydrolysable tannins group formed by a unit of sugar and a variable number of phenol acid molecules. Its distribution covers different families of the vegetable Kingdom, as Anacardiaceae, Fabaceae and Myrtaceae (BATTESTIN et al., 2004; SANTOS; DE MELLO, 2010) as well as in fungi of the genus Termitomyces (PUTTARAJU et al., 2006).
Carl Wilhelm Scheele scientist was the first to identify the gallic acid in plants in 1786 (FISCHER, 1914). However this molecule is arousing the interest of researchers mainly for its antioxidant capacity (KIM et al., 2007). Other pharmacological activities are described in the literature as anticancer (CHIA et al, 2010; LIANG et al., 2012), HIV (KRATZ et al., 2008), antiulcerogenic (JUNG et al., 2013), anti-inflammatory (CHOWDHURY et al., 2013), antimicrobial (KUBO et al., 2003), antifungal (KUBO et al., 2001) among others. Recently, some studies have been published relating the effect of gallic acid before the formation of amyloid plaques, considered to be the initial step in Alzheimer’s disease (LIU et al., 2013; JAYAMANI et al., 2014; LIU et al., 2014).
In addition to the medicinal aspects, gallic acid is applied in different areas. Its first applicability was on skin and leather industry, as a chelating agent (COSTA et al., 2013). The first photographs had used gallic acid as a developer (BENIWAL et al., 2013). Gallic acid is used for the synthesis of trimethopim, an antimicrobial agent, and also as a preservative in food and beverages, primarily by its power to kidnap to free radicals (BAJPAI & PATIL, 2008).

2. Chemical Aspects, biogenesis and synthetic production

Gallic acid (3,4,5 trihydroxybenzoin acid) is a crystalline solid, slightly colourless or slightly yellow. Its molecular weight is 170.11954 g/mol and its molecular formula is C7H6O5. The melting point is 210°C with decomposition between 235 to 240°C, producing carbon dioxide and carbon monoxide. Its density is 1.69 kg/L (20°C), its pKa is 4.40 and the Log P of 0.70 (20°C). It is soluble in water, alcohol, ether and glycerol and practically insoluble in benzene, chloroform and ether petroleum (PUBCHEM, 2015). The chemical structure of gallic acid is shown in Figure 1.
Although it has not defined completely its biogenesis, it is known that it has its origin in the shikimic acid pathway, an important route in the production of secondary metabolites of aromatic structure, existing in vegetables and certain microorganisms. This route starts with some amino acids, in particular L-phenylalanine, and produces important class of compounds as coumarins, alkaloids, lignans and polyphenols (DEWICK, 1969).
Three possible routes are described for its production (Figure 2). The first suggests an initial conversion of phenylalanine in caffeic acid, then in acid 3,4,5 trihydroxycinnamic and finally in gallic acid. Another route suggests that comes directly from the production of the acid 3,4,5 trihydroxycinnamic, considering this was never found in nature. Thus, the production of side chain occurs by the formation of protocatechuic acid, from caffeic acid (SANTOS; DE MELLO, 2010). A third route suggests that, from the shikimic acid by the action of dehydrogenase, occurs to the formation of 3-dehydroshikimic acid. This follows from a spontaneous aromatization, resulting in the production of gallic acid (KAMBOURAKIS et al., 2000).

On an industrial scale, gallic acid is produced by the breakdown of tannic acid by tanase, a glycoprotein esterase. This enzyme produced by different microorganisms in particular fungi as the genera Aspergillus and Penicillium, mostly by the fermentation process (BELMARES et al., 2004; MACEDO et al., 2005). Costa et al. (2013) obtained tanase and gallic acid from Aspergillus tamarii in solid medium and submerged. This species has also been presented better result, along with Aspergillus japonicus in the studies of Melo et al. (2013), in search of tanases- producing fungi in caves of the Brazilian regions, caatinga and cerrado.
3. Analytical methods

Among the different analytical methods include spectroscopy, chromatography and capillary electrophoresis. Other techniques are also used as thermal analysis and the chemiluminescence analyses.
3.1. Spectroscopy

Gallic acid offers maximum absorption at 272.5 nm with log ε 4.06 (PUBCHEM, 2015). This absorption is a result of the sum of the absorptions of the main chromophore group (cluster benzoil), added the three substitutions-OH (two goal and one for), which theoretical calculations result in absorption of 269 nm (PAVIA et al., 2013). However, it is possible to observe slight variations around that value. Pawar & Salunkhe (2013) using UV-Vis spectrophotometry, quantified and validated method for gallic acid in hydroalcoholic extract of Thipala churna in 273 nm. Vijayalakshmi & Ravindhran (2012) obtained fingerprints of Diospyrus ferrea Willd., obtained in different solvents, in which verified that the maximum absorption of gallic acid occurs between 270 and 275 nm, with better results for the ethanolic extract.

The great part of the application of spectroscopy in the UV-Vis is in the determination of total polyphenols. Folin-Ciocalteau’s method (FC) is a colorimetric method based on transfer of electrons between reagents and polyphenols. In this essay, gallic acid is used as the default and the results expressed as gallic acid equivalents (STALIKAS, 2007; SÁNCHEZ-RANGEL et al., 2013; BALAN et al., 2015). Chaves et al. (2013) developed and validated method for Guapira graciliflora and Pseudobombax marginatum, aimed at evaluating the production of metabolites during periods of rain and drought. A variation of this technique is the use of the Folin-Denis, which was an improvement over the previous method (MONTEIRO et al., 2005).
Another possibility of quantification for the total polyphenols was using near-infrared spectroscopy (NIR), which corresponds to the spectrum between 4000 to 10,000 cm1. However, for the best viewing and interpretation of data, chemometrics tools were applied as the minimum partial squares (PLS) or regression in core components (CRP) (CHEN et al., 2006; MONCADA et al., 2013). The NIR was used in the prediction of total polyphenols in green tea (Camellia simensis L.), also using the PLS when comparing the data obtained with HPLC, the method using NIR proved faster, economical and allowed for the simultaneous quantification of different compounds (SCHULZ et al., 1999).
In infrared spectroscopy, gallic acid presents specific regions of absorption. Vijayalakshmi & Ravindhran (2012) identified eight major peaks included in 1022, 1234, 1448, 1622, 1714, 3043, 3280 and 3365 cm1, being that the peak in 1714 cm1 is considered the most important because it is specific to polyphenols. On the other hand, Lam and collaborators (2012) found as characteristic peaks: 3492, 3370 and 3282 cm1 (related to different forms of hydroxyl); in 2920

and 2850 cm1 corresponds to the stretch of C-H in aromatics; in 1701 cm1 corresponds to the carbonyl absorption in carboxylic acid, 1615 cm1 stretch related carbon-carbon in alkenes.
3.2. Chromatography

Chromatographic methods are the most used for identification and quantification of gallic acid in different matrices. Among the methods include thin layer chromatography, gas chromatography and high performance liquid chromatography (HPLC).
3.2.1. Thin layer chromatography (TLC)

The TLC is widely used in order to be a fast, low-cost method that allows a first “fingerprint” on the chemical composition of extracts of medicinal plants (SONOWANE et al.., 2012; TÓTOLI & SALGADO, 2014). As it is a technique in which the separation stems from migration of compounds against the stationary and mobile phases affinity, perhaps the most crucial step in the development of methods for TLC it’s your optimization (PEDROSO & SALGADO, 2014).
Sharma et al. (1998) used the TLC identification of gallic acid and other phenolic compounds, using silica gel plates. Among all stages tested mobile phase consisting of chloroform: ethyl acetate: acetic acid (50: 50: 1) was that best separated compounds. For the revelation of the plates, we used the ferric chloride and sulphuric acid, vanillin. Gallic acid was also identified in Schinus terebinthifolius Raddi and Arctostaphylos uva-ursi (L.) Spreng. In both species the mobile phase used was toluene: ethyl acetate: methanol: formic acid (75: 25: 10: 6) and as revealing 1% FeCl2 (BRAZ et al., 2012).
Dhralwal et al. (2008) have developed and validated a method of quantification in Bergenia ciliata and Bergenia ligulata by TLC. The chosen mobile phase was toluene: ethyl acetate:

formic acid (40: 60: 10). However, instead of a revealing solution, reading was made using an automated system called HPTLC (High Performance Thin Layer Chromatography) or TLC densitometric. This technique was also used for quantification in Hygrophila auriculata (K. Schum) (HUSSAIN et al., 2012), Terminalia chebula (KUMAR et al., 2010), Acacia leucophloea (LEELA et al., 2013) and Syzygium aromaticum (L.) Merr. & Perry (PATHAK et al., 2004). The main difference among these works is the variation of the composition of the mobile phase. However, in all of it is remarkable the presence of an acid (glacial acetic acid or formic acid), causing the pH of the stage more acid. Thus, there is a suppression of ion for the gallic acid (ARAPITSAS, 2012).
3.2.2. Gas Chromatography (GC)

Although gas chromatography is a technique which allows the identification and quantification of various compounds, GC analysis requires that the compound to present a good volatility and which does not boil above 300°C, such as essential oils, or volatilisable. Moreover, it is a technique that has high sensitivity and delectability (PENTEADO et al., 2008).
In the case of gallic acid, is possible the sample derivatization. This procedure allows a better response of the detector front of the chromatographic system, allowing its analysis by GC (FRIAS et al., 2014). Tor et al. (1996) when determining gallic acid and pirrogalol in biological matrices by gas chromatography coupled to mass spectrometry (GC/MS) derivatization the sample prior to injection. It as used a column J & W Scientific® 15 m X 0.53 mm X 0.1 µm DB- 1, helium gas drag and flow rate of 7 mL/min, operating at 60°C and oven temperature gradient system up to 275°C. The sample derivatization was also adopted by Kuskoski et al., (2012) by analyzing phenolic compounds by GC/MS Ion Trap in guarana (Paullinia cupana). To this end,

the research used a fused silica capillary column (Phenomenex ® Zebron ZB-5ms 30 m X 0.25 mm X 0.25 mm), with a temperature of 300°C injection and helium gas drag with flow rate of 1 mL/min.
Phenolic compounds, free sugars and polyols have been determined in mango (Mangifera indica L.) by means of a GC/MS, with sample derivatization. Initially the sample was diluted in pyridine. Then it was sylilated to 80°C for 30 minutes in N, O-bis-(trimethylsilyl)- trifluoroacetamida (BSTFA) containing 1% of trimethilclorosilane (TMS) as a catalyst. The derived TMS was kept in iso-octane prior to analysis. The research used a column DB1 J & W, 25 m X 0.2 mm X 0.33 µm, with helium as drag gas (flow of 1 µL/min) and oven temperature gradient system up to 290°C (NUNES SELLÉS et al., 2002).
3.2.3. High performance liquid chromatography

All the techniques, the high performance liquid chromatography (HPLC) is the most applied in the identification and quantification of gallic acid. Its great power and its resolution better separation among the compounds made of this technique the gold standard in the analysis of gallic acid (STALIKAS et al., 2007). As the phenolic compounds present high molecular weight and high polarity, the use of a technique of separation becomes applicable, considering this compound be the training framework of hydrolysable tannins (ARAPTISAS, 2012).
Another great advantage of liquid chromatography is the application in different arrays, mainly herbal extracts. Thus, the correct choice of the method and solvent extraction is the first critical point analysis of gallic acid by HPLC. Different techniques such as turbo-extraction, ultrasonication, maceration and infusion can be used, being these last two the most used (Table 1). Generally, the extraction methods can be classified into conventional (reflux, infusion and

steeping, for example) and non-conventional or modern (turbo-extraction and ultrasonication). However, there is not yet a general method, making necessary the use of optimizations in the extractive process (BRUSOTTI et al., 2014).
One of the solvents used, hydroalcoholic solutions are the most applied, although the methanol, acetone and water may also be used. In the case of water, its application is mainly on hot or cold infusion, mainly focused on production of teas. According to Azmir et al. (2013) methanol and ethanol are the most suitable solvents for extraction of phenolic compounds. Since acetone is more suitable for extraction of flavonoids, while water is more used in the extraction of tannins.
As regards the chromatographic columns most of the work using a reversed-phase column (C-18 and C-8). The most applied were the columns of 250 mm. However are noticeable smaller columns as 150 mm and 100 mm in the analysis by UPLC. Although the time of analysis with the use of larger columns is a little bigger, its use is justified due to the large number of compounds present in the array (HE et al., 2015).
Stationary reverse phases allow the retention of ionizable compounds, in case the gallic acid, since supress their ionization. Thus, it is necessary the use of a mobile phase ion-par, aiming to maintain the pH of mobile phase just below the pKa of gallic acid (LEE et al., 2009; RÓSES et al., 2009). As mobile phase acidifying was employed the formic acid, TFA, and acetic acid orthophosphoric (table 1). De Souza (2002) suggests that for the analysis with detection in the UV-Vis, orthophosphoric acid is the most suitable, due to good resolution in the chromatogram. Since the formic acid, in addition to reducing the effect of tail of the chromatogram, allows an increase in ionization in the case of MS/MS (MAHFOUDHI et al., 2014). In some cases it is

necessary to use the acidification of two phases (aqueous and organic) due to structural similarities of the compounds (WANG et al., 2000).
The organic phase modifiers complement the mobile phase used in analyses by HPLC. The most used are methanol and acetonitrile (Table 1), usually in gradient system with the aqueous phase. Though do not upgrade a column at the time of protection analysis, acetonitrile promotes a better resolution of the peaks (STAKILAS et al., 2007).
About the detection system, UV-Vis/Diode array detector (DAD) is undoubtedly the most widely used, although the mass spectroscopy can be used, mainly in pharmacokinetic analyses (SUN et al., 2014) and UPLC (Table 1). One of the wavelengths, the region between 270-280 nm is the most applied. Some works use 210 nm, for being related to the primary absorption of gallic acid (WANG et al., 2000), or 254 nm of general absorption of phenolic compounds (ARCEUSZ et al., 2013).
Therefore, after the optimization of the parameters of analysis (extraction, mobile phase, stationary phase and detection) it is possible to obtain different retention times. It is possible to obtain times and retention among 1.69 minutes until 29 minutes for the HPLC and UPLC until 2 minutes (Table 1). The great difference between the retention times can be related to gradient system used in some analysis, as the work performed by Choi et al. (2011).
3.3. Capillary electrophoresis

Although it is a technique still little natural products, the capillary electrophoresis and its related techniques (CZE-capillary zone electrophoresis, MEKC-micellar eletrokinetic chromatography, CEC — capillary eletrokinetic chromatography) aims to be an excellent green alternative for identification of these compounds, mainly gallic acid (GOTTI., 2011). The

technique consists in the separation of different ionizable compounds by difference of migration front of an electric field. The first time this technique has been applied, dates from 1930 by the Swedish chemist Arne Tiselius (SPUDEIT et al., 2012).
Cartoni et al. (1996) used the capillary electrophoresis in separation of gallic acid and other phenolic compounds in Italian wines and other alcoholic beverages. To this aim, they used a fused silica capillary with 43 cm long, in hydrodynamic method at 25°C and UV-Vis detector. After several tests, the best pH for analysis was 8.3, with a potential of 15 kV using sodium bicarbonate buffer at 50 mM. Wu et al. (1996) applied the CZE and MEKC on separation of components of Paeoniae radix, a formulation for use in traditional Chinese medicine composed by dried roots of Paeonia veitchii Lynch or Paeonia lactiflora Pall, using a buffer solution Na2B4O7.
Using the micellar eletrokinetic chromatography (MEKC), Prasongsidh & Skurry (1998) developed a method to analyze the gallic acid and other phenolic compounds (reverastrol and quercetin, catechin) in wines. They used a fused silica capillary of 64.5 cm, and obtained a good separation between the compounds by up to 11 minutes. Vaher et al. (2003) produced a similar work, however in plant extracts (Myrica gale L., Hippophae rhamnoides L., Rosa majalis L. and Reynutria japonica Houtt.). Using capillary electrophoresis coupled to a UV-Vis detector in 240 nm, an uncoated capillary of 75 cm and as a buffer with sodium tetraborate pH 9.4 and concentration of 25 mM. Another study determined the gallic acid and salidroside in Rhodiola dumulosa and medicinal preparations. Once again, the pH of the buffer solution was fairly alkaline (between 8 and 11), aiming at improving the ionization of the compounds. A column of

fused silica was used at 25°C with voltage of 15 kV, obtaining good results in the separation of compounds (YUE et al., 2006).
3.4. Other techniques

The evaluation of the solubility is an important physical-chemical parameter. Daneshfar et al. (2008) assessed the solubility of gallic acid at different temperatures (between 25 and 60°C) and in ethyl acetate, water, ethanol and methanol; the latter presented better solubility. For both, the authors used UV-Vis spectrophotometry (269, 269.5, 273 and 273.5 nm) for quantification.
The chemiluminescence analyses were also applied by Li et al. (2012) for quantification of gallic acid. We used the luminol-AgNO3-Ag for complexation and reading in a spectrophotometric flow system in the region of the UV-vis. This was possible due to oxidation caused by gallic acid reagent, allowing its detection.
Thermal analysis, in particular the differential scanning calorimetry (DSC) and thermogravimetry (TG), can also be applied to the identification of compounds and potential degradations and herbal drugs (MANN et al., 200; FERNANDES et al., 2013). When performing the DSC curve of gallic acid in a ratio of heating of 10°C/min in N2 atmosphere, Shyam et al. (2012) achieved start temperature melting point (T on set) at 136.5°C, which may suggest that the sample was not pure or the occurrence of polymorphism, due to the gallic acid melt in 210°C. Guo (2012) to perform analysis thermogravimetric in different rate of heating (5, 10 and 15°C/min) obtained three stages of degradation. The first one (± 65°C) would be related to loss of oxygen of the hydroxyl at position orto, the second (± 135°C) the production of CO2 and the third, to complete degradation from 305°C.

4. Conclusion

It is notorious the biological activity of gallic acid, either as isolated molecule, either as a constituent of vegetable matrices. In this way, different analytical methods are needed for identification and quantification of this active ingredient in various biological matrices.
Although some methods such as liquid chromatography, are well consolidated innovative vision is important for green methods such as capillary electrophoresis and infrared spectroscopy and little exploited as methods for thermal analysis. The approaches described in this work can be useful in the evaluation of gallic acid from raw materials such as vegetable drug, until the final product, such as teas and capsules.

The authors acknowledge CNPq (Brasília, Brazil) and CAPES Foundation.


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Table 1 — Instrumental conditions used in liquid chromatography for the determination and quantification of gallic acid.

Sample preparati on
Chromatog raphy condition
Reten tion time
Refere nce
Camellia sinensis Infusion The sample added to boiling water.
After cooling, the sample was filtrated, diluted and frozen
until use. LiChroC ART 250-4
LiChros pher 100 RP-8 (2504
mm, 5 µm) Gradient — A:
trifluoroacet ic acid 0.05%; B:
methanol. Flow rate:
0.8 ml/min. DAD 7.89 Gil et al., 2011
Camellia Ethanol Extraction LiChroC Isocratic — UV-Vis 3.23 Karam

sinensis, Arctostaph ylos uva- ursi, Corylus avellana, Oenothera biennis, Vitis
vinifera and acetone extract with ethanol 95% and acetone 80% ART 250-4
LiChros pher 100 RP-8 (2504
mm, 5 µm) water: acetonitrile: acetic acid (88:10:2).
Flow rate:

1.0 ml/min (280 and

350 nm) ac et al., 2006
Cerasus avium Maceratio n with shaking Extraction with water and ethanol Machere y-Nagel Nucleod er C18 Gravity column (125 × 2
mm, 5

µm) Gradient — A: methanol with formic acid 0.5%; B: formic acid 0.5%. Flow rate:
0.3 ml/min. MS/MS

(Tandem Gold triple quadripole) 2.50* Bursal et al., 2013
Green tea Infusion Hot water extraction Kingsorb C-18 Isocratic — methanol/ UV-Vis (210 nm) 2.60 Wang et al.,

(150 ×

4.6 mm, 5 µm) water/ phosphoric acid. Flow rate: 1.0
ml/min 2000
Chinese Hamster Ovary (CHO) cell line Cell lysate Centrifuga tion Gemini C-18
Phenome nex (1002
mm, 5 µm) Isocratic — formic acid 0.05% and acetonitrile. Flow rate:
0.35 ml/min MS/MS

(hybrid triple quadrupole/ linear ion trap mass spectromet
er) 0.98 Wang et al., 2013
Dendropht hoe falcate Raw material Extraction with methanol Thermo MOS 2
Hypersil C18
column (250 cm
× 4.6

mm, Isocratic — acetonitrile and water with 0.1% phosphoric acid (60:40%).
Flow rate: UV-Vis (271 nm) 12.81 Deshm ukh & Prabhu
, 2011


ODS 3) 1.0 ml/min
Emblica officinale and Glycyrrhiz a glabra Hommade Syrup Dilution simple in methanol- water (1:1). Inerstil ODS C- 18 (4.6250
mm, 5µm) Gradient — A:
acetonitrile; B: water with acid water (0.1% phosphoric acid). Flow rate; 1.0
ml/min. UV-Vis (251 nm) NA Deodh ar et al., 2011
Hamamelis virginiana Sonicatio n Extraction with water in ultrassoun d Kingsorb C-18 (150 ×
4.6 mm, 5 µm) Gradient — A:
phosphoric acid 0.1%; B: methanol and phosphoric acid 0.1%.
Flow rate: UV-Vis (210 nm) 3.00* Wang et al., 2003

1.0 ml/min.
Ilex paraguarie nsis Infision and maceratio n Hot infusion; Hydroetha nolic extraction
(70%). Shimadz u RP C8
(4.5 mm

× 150
mm, 5 µm) Isocratic – methanol/w ater. Flow rate: 0.5 ml/min. DAD (280

nm) 17.00

* Pereira et al., 2012
Juglans mandshuri cae Sonicatio n Hydroetha nolic extraction (74%)
with ultrassoun d Venusil ASB C18 (100
× 2.1

mm, 5 µm) Gradient — A: formic acid 0.1%; B:
Acetonitrile. Flow rate:
0.2 ml/min. UHPLC MS/MS
(Triple- quadrupole tandem mass spectrometr
y) 1.80* Sun et al., 2014
Labisa pumila Methanoli c extract Dried leaves were extracted
with Intersil GL
Science ODS-3
(5 μm Gradiente– A: water with trifluoroacet
ic acid (pH UV-Vis (280 nm) 4.00* Karimi et al., 2011

methanol 80% and added HCl. The extract was dried and resuspend ed in
methanol. 4.6 × 150 mm) 2.5); B:

acetonitrile. Flow rate:
0.6 ml/min.
Michelia alba, Caesalpini a pulcherrim a, Nelumbo nucifera Ethanolic extract Extraction with ethanl 95% by ultrassoun d and dilution in mobile
phase. Luna C18 RP (4.6250
mm, 5 µm) Gradient — A;
water/acetic acid (25/1) e B:
methanol. Flow rate:
1.0 ml/min. UV-Vis (280 nm) 5.00 Samee & Vorara t, 2007
Nymphaea stellata Soxhlet extraction Hydroetha nolic HiQ Sil C-18 Isocratic: phosphoric UV-Vis (265 nm) 6.50 Rakes h et

extract (70%) (250 ×

4.6 mm, 5 µm) acid 0.01%/ acetonitrile. Flow rate:
1.0 ml/min. al., 2010
Phyllanthu s emblica Fresh fruit juice The juice was dried by freezer- dried. It was diluted with methanol:
water Zorbax SB RP- C18, 2504.6
mm and 5 µm Gradient — A: acetic acid 0.01%; B:
methanol. Flow rate:
0.9 ml/min. DAD (279

nm) 10.77 Sawan t et al., 2011
Phyllanthu s niruri Dried extract Roots, backs and leaves were extracted by aqueous
decoction RP-18

LiChros pher 2504
mm e 5 μm Gradient — A:
phosphoric acid 1%; B; acetonitrile with phosphoric
acid 1%. UV-Vis (275 nm) 5.00* Couto et al., 2013

by reflux

and dried. Flow rate:

0.6 mL/min.
Psidium guajava Maceratio n Aqueous extraction and lyophilizat ion Gemini NX C-18
Phenome nex (2504.6
mm, 5 µm) Gradient — A:
phosphoric acid 0,5%; B.
acetonitrile/ phosphoric acid 0.5%. Flow rate:
0.8 ml/min DAD (280

and 352 nm) 9.00* Verza et al., 2007
Punica granatum Hydroalco holic extract of bark Diluted extract in methanol: water (60:40%) Shimadz u ODS C-18 (4.6250
mm, 5µm) Gradient — A: water with formic acid 1%; B: acetonitrile. Flow rate:
1.0 ml/min UV-Vis (254 nm) 29.00* Choi et al., 2011
Punica Juices, All Kinetex Gradient/iso DAD (270 2.45 Qu et

granatum drinks and aqueous extracts. simples were diluted in distilled water. 2.6 µm C-18 4.6100 mm cratic — A: phosphoric acid 0.01%; B:
acetonitrile with phosphoric acid 0.01%. Flow rate:
1.8 ml/min. nm) al., 2012
Rhizophora apiculata Purified extracts containin g hydrolisa ble and condesed
tannins. Purified fractions by TLC Intersil ODS
Science Inc (10 μm, 4.6300
mm) Isocratic — methanol and water (70:30%).
Flow rate:

1.0 ml/min. UV-Vis 2.96 Hong et al., 2011
Schinus terebinthifo lius Raddi Ethanol extract (EE), EE:
maceratio n; EH: RP 18

ACE 121

1503 (5 Gradient: A: acetonitrile: water; DAD 3.19
and 1.69 Carval ho et al.,

hydrolise d extract (HE) and gel. extract with reflux and sulfuric acid Gel: solubilizat ion of all compound
s. μm, 2504.6
mm, 100 A) B:methanol: water with formic acid to pH 2.7.
Flow rate: 1.0
ml/min.** 2006
Scutia buxifolia Infusion Aqueous extraction and lyophilizat ion Shimadz u (250 ×
4.6 mm, 5 µm) Gradient — A: acetic acid 2%; B: Methanol. Flow rate:
0.7 ml/min. DAD (254

nm) 17.00* Freitas et al., 2013
Stryphnode ndron adstringens

Stryphnode ndron Extract with acetone:w ater (7:3) Turboextr action (20 min). The extract was concentre Gemini NX C-18
Phenome nex (2504.6
mm, 5 Gradient — A: water/ trifluoroacet ic acid 0.05%; B :
acetonitril/ UV-Vis (210 nm) 10.00 Lopes et al., 2009

polyphyllu m, Stryphnode ndron
obovatum d, dried and partitioned
. µm) trifluoroacet ic acid 0.05%.
Flow rate:

0.8 mL/min.
Thymus vulgaris, Salvia officinalis, Origanum majorana Maceratio n Extract with methanol Zorbax SB-C18 (250 ×
4.6 mm, 5 µm) Gradient — A: formic acid 0.05% and B: methanol. Flow rate:
0.8 mL/min UV-Vis (280 nm) 5.85* Roby et al., 201)
Triphala churna Maceratio n Extract with methanol Phenome nex (250
× 4.6

mm, 5 µm) Gradient — A;
acetonitrile and B: a phosphoric acid 0.1%. Flow rate:
0.8 mL/min. DAD (254

nm) 5.29* Platel Madha vi et al., 2010