A Study of Polyphenolic Compounds and in vitro Antioxidant Activity of Trianthema portulacastrum Linn. Extracts

Antioxidants are chemical substances derived from natural or synthetic origin, which protect the body from oxidative stress. Studies suggest that antioxidants possess anti-inflammatory, antimutagenic and anti-carcinogenic activities (Hamid et al., 2010).

Trianthema portulacastrum Linn. (Aizoaceae family) commonly known as horse purslane is an exotic plant growing as ‘weed’ in Africa, Southeast Asia and tropical America. The plant has been reported to possess a variety of pharmacological actions, including analgesic, antipyretic, antioxidant, anti-inflammatory, hypolipidemic, hypoglycaemic, antibacterial, antifungal and anticancerous activities.

Polyphenolic compounds are important antioxidants which exhibit chemopreventive actions like antioxidant, anticancer, antimutagenic and anti-infiammatory effects (Huang et al., 2010). Polyphenol rich foods and beverages help to increase plasma antioxidant capacity, hence, consumption of antioxidants is associated with reduced levels of oxidative damage. Oxidative damage to cells is the main pathological disturbance of numerous chronic diseases. Minimizing oxidative damage can be one of the most important approaches to the primary prevention of chronic diseases and ageing-associated health problems. Trianthema portulacastrum Linn. displays excellent oxidative stability suggesting the possible presence of phenolic compounds that act as antioxidants.

The present study was aimed to determine and quantify the polyphenolic compounds present in aqueous, ethanolic, hydroethanolic, methanolic and hydromethanolic extracts of Trianthema portulacastrum using high performance liquid chromatography (HPLC), to identify the unknown compounds by gas chromatography- mass spectrometry (GCMS) and to estimate the antioxidant properties of different extracts by various methods.

Plant material and preparation of extracts
 
The aerial parts of T. portulacastrum were collected before flowering stage from nearby fields of Pantnagar, Uttarakhand during the months of January to April 2016. The plant was taxonomically identified and authenticated by the Botanical Survey of India (BSI), Dehradun and the voucher specimen of the plant has been deposited in herbarium of BSI, Dehradun with Accession No. 116105. The study was conducted in the Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture and Technology, Pantnagar.

Five extracts viz. aqueous, ethanolic, hydroethanolic, methanolic and hydromethanolic were prepared by extraction in water, ethanol, hydroethanol (1:1), methanol and hydromethanol (1:1), respectively. The extracts were prepared by cold maceration method (Handa et al., 2008). The dried extracts were kept in air tight glass containers in deep freeze at -20°C.
 
Determination of polyphenolic profile of plant extracts by HPLC
 
A modified method (Zhang et al., 2013) was standardized for detection and quantification of 16 polyphenolic and flavonoid compounds present in plant extracts in a single run. HPLC System (Shimadzu Corporation, Japan) consisting of binary pumps along with UV detector was used. Reverse phase C18 column along with guard column were used. Column temperature was set at 25°C. Loop size of 20 µl was used. Mobile phase consisted of A (3% acetic acid) and B (acetonitrile) with gradient elution at total flow rate of 0.5 ml/min. Gradient time programme for pumps consisted of 0% B up to 11.5 min, 5% B from 11.5 to 19.5 min, 10% B from 19.5 to 32.5 min, 19% B from 32.5 to 45.0 min and 37% B from 45.0 to 60.0 min. Total run time was 60 min.

Pure analytical standards of polyphenolic and flavonoid compounds purchased from Sigma Aldrich, USA were used as reference standards. Working standard solutions (0.001-10 µg.ml^-1) were prepared in mobile phase A. The dissolved plant extracts were filtered through 0.20 µm filters (Millex, 13 mm, nylon) before direct injection into the HPLC system.
 
Chemical fingerprinting of plant extracts using gas chromatography mass spectrometry (GCMS)
 
Gas chromatograph mass spectrometer, model GCMS-QP2010 Ultra (Shimadzu Corporation, Japan) equipped with autosampler, autoinjector and Supelco SP-2560 fused silica capillary column was used for the study. The software GC-MS solution ver. 4 was used to analyze mass spectra and chromatograms. The results of mass spectra were compared by making similarity search using NIST11.lib and Wiley8.lib spectral library search programs linked with GC-MS instrument.
 
Determination of total phenols
 
Total phenols in plant extracts were quantified using Folin-Ciocalteau reagent method (Singleton and Rossi, 1965) with modifications.
 
Determination of total flavonoids
 
Total flavonoids in plant extracts were quantified by aluminum chloride colorimetric method (Zhishen et al., 1999) with modifications.
 
DPPH radical scavenging assay  
 
DPPH Radical Scavenging Assay was assessed according to the previously described method (Burits and Bucar, 2000) with some modifications.
 
ABTS radical cation (ABTS•+) scavenging assay
 
The assay was performed as per the previously described method (Re et al., 1999) with some modifications.
 
Total antioxidant capacity by phosphomolybdenum method
 
The total antioxidant capacity (TAC) of plant extracts were evaluated according to a previously described method (Prieto et al., 1999).
 
Statistical analysis
 
Results of the study were analyzed applying one way ANOVA and expressed as Mean±SEM. The differences were considered statistically significant at p<0.05.
 

Fresh plant materials of Trianthema portulacastrum after complete drying at 37°C yielded 11.71% dry matter. There was wide variation in percent yield of different extracts ranging from 2.96%, 7.49%, 12.53%, 16.01% and 20.88% as in case of TPE, TPM, TPA, TPHM and TPHE, respectively. It may be due to varying degree of solubility of phytochemicals present in plants extracted in different polarity solvents used in the extraction process. As per a previous study, phytochemical composition, antioxidant and reducing activities of the extracts are positively associated with the use of organic solvents during the extraction process (Ganguli et al., 2018). In our study, the higher yield of hydroethanolic and hydromethanolic extracts supports the findings.
 
Determination of polyphenolic profile of plant extracts by HPLC
 
All 16 compounds were eluted within a run time of 60 min in gradient flow (Fig 1).



The retention time (RT), coefficient correlation (R2), limit of detection (LOD) and maximum absorbance wavelength (λmax) have been presented in Table 1.



The standardized method for determination of polyphenolic compounds presents good validation parameters like linearity (R2, 0.999), precision (consistent RT), range and LOD.

Gallic acid, protocatechuic acid, catechin hydrate, vanillic acid, epicatechin, p-coumaric acid, rutin, salicylic acid, myricetin, quercetin and trans-cinnamic acid were estimated to be present in plant extracts being maximum in hydroethanolic followed by aqueous, hydromethanolic, ethanolic and then methanolic extracts. Catechin, epicatechin, rutin, myricetin and quercetin were main flavonoid compounds in most of the plant extracts.

Apart from identified peaks (Fig 2), a good number of peaks were detected with reasonable peak areas indicating presence of a large number of other unknown phytochemicals too, in extracts.



Hydroethanolic extract of T. portulacastrum contained 1231.97 µg catechin, 245.18 µg epicatechin, 683.62 µg rutin, 1992.14 µg myricetin and 165.37 µg quercetin per gram of extract which was highest among other extracts. A previous study (Al Sherif and Gharieb, 2011) reported para-hydroxybenzoic, vanillic, ferulic, o-coumaric, pyrogallic, protocatechuic and trans-cinnamic acids in methanolic extract of T. portulacastrum which is in accordance with the findings of our study. Another study (Jabbar et al., 2019) supports our findings who reported the presence of five important compounds including caffeic acid (3.17 ppm), gallic acid (3.22 ppm), quercetin (4.11 ppm), cinnamic acid (11.81 ppm) and chlorogenic acid (16.11 ppm) in 70% ethanolic extract of T. portulacastrum.

Polyphenolic and flavonoid compounds have been reported to possess antioxidant properties which can help in ameliorating oxidative stress (Ganguli et al., 2018). Plant derived phenolic compounds are safer and promising sources of antioxidants which can be utilized for therapy of various diseases. Trianthema portulacastrum Linn. displays excellent oxidative stability suggesting the possible presence of phenolic compounds that act as antioxidants.
 
Chemical fingerprinting of plant extracts using GCMS
 
The GCMS mass spectral analysis of ethanolic and methanolic extracts of T. portulacastrum (Fig 3, 4) revealed the presence of various bioactive compounds like β-sitosterol, stigmasterol, squalene, n-hexadecanoic acid, hexadecanoic acid trimethylsilyl ester, 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-, oleic acid, octadecanoic acid, mome inositol, phytol and cis-9-hexadecenal.





Phytosterols are potent antioxidants. These directly inhibit tumor growth by slowing cell cycle progression, inhibition of tumor metastasis and induction of apoptosis (Bradford and Awad, 2007). Squalene, a triterpene which is a key intermediate in the synthesis of plant and animal steroids, possesses antioxidant, chemopreventive activity against colon carcinogenesis (Rao et al., 1998) and skin cancer. Dodecanoic acid, tetradecanoic acid, n-pentadecanoic acid, 9,12-octadecadienoic acid (z,z)- and n-hexadecanoic acid (synonym: palmitic acid) have antioxidant and antimicrobial activities. 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- (synonym: α-linolenic acid) which was present in all the extracts in very high amounts is a proven hypocholestaemic agent which reduces the risk of cardiovascular diseases. Studies regarding GCMS spectral analysis of various extracts of T. portulacastrum are very less.
 
Determination of total phenols and flavonoids of T. portulacastrum extracts
 
The total phenol content ranged from 54.43±3.14 to 106.51±3.09 mg GAE/g extract with lowest in aqueous and highest in hydroethanolic extract. Hydromethanolic, methanolic and ethanolic extracts also exhibited significant phenol content. The total flavonoid content varied from 4.24±0.33 to 20.93 mg rutin equivalent/g extract. Aqueous extract exhibited significantly (p<0.05) lower content, moderate in ethanolic and methanolic with highest in hydroethanolic extract.

Several phenolic compounds identified in our study have been reported to possess strong antioxidant activity. Gallic acid induces apoptosis which is associated with ROS mediated oxidative stress, mitochondrial dysfunction and an increased intracellular Ca²+ level (Inoue et al., 2000). It is a powerful antioxidant and has been considered a useful phytochemical for cancer chemoprevention. It is used as a reference standard for determining total phenol content in plant extracts and other analytes. Protocatechuic acid, vanillic acid, catechins, rutin, quercetin and myricetin have been reported to contain excellent antioxidant properties. Majority of the flavonoids exhibit strong antioxidant activity (Al Sherif and Gharieb, 2011).
 
DPPH radical scavenging assay of plant extracts
 
DPPH radical scavenging activity of different extracts of T. portulacastrum expressed in terms of ascorbic acid equivalents has been presented as Table 2.



The antioxidant activity measured in ascorbic acid equivalents for TPA, TPHE, TPHM, TPE and TPM extracts were found to be 44.45±0.58, 92.68±2.65, 54.64±0.84, 25.52±0.90 and 39.26±0.79 mg AAE/ g of extract, respectively, which differed significantly (p<0.05) among themselves.
 
ABTS•+ scavenging assay of plant extracts
 
ABTS•+ scavenging activity of different extracts of T. portulacastrum expressed in terms of ascorbic acid equivalents has been presented as Table 2. IC50 value was found to be 17.06±0.35 µg/ml for ascorbic acid. IC50 values for TPA, TPHE, TPHM, TPE and TPM extracts were found to be 122.16±2.12, 76.97±1.16, 87.29±0.70, 190.51±5.39 and 131.18±1.74 µg/ml, respectively, which differed significantly (p<0.05) among themselves. Lowest IC50 value of 76.97±1.16 µg/ml was obtained for TPHE whereas highest was for TPE (190.51±5.39 µg/ml) extract. AAE for TPA, TPHE, TPHM, TPE and TPM extracts were found to be 139.82±5.29, 221.77±6.64, 195.50±5.44, 89.60±1.41 and 130.10±3.26 mg AAE/g extract.
 
Total antioxidant capacity (TAC) of plant extracts
 
Total antioxidant capacity (TAC) determined by phosphomolybdenum method for different extracts of T. portulacastrum has been presented in Table 2. The antioxidant capacity was expressed in terms of milligram of ascorbic acid equivalent (AAE) per gram of extract.

TAC for various extracts of T. portulacastrum ranged from 56.79±2.94 to 103.51±4.50 mg AAE/g extract with highest value in hydroethanolic and least in aqueous extract. Hydromethanolic, ethanolic and methanolic extracts accounted moderate activity of 85.84±3.35, 61.39±2.22 and 57.61±1.83 mg AAE/g extract, respectively.

There is direct relationship of AAE with the antioxidant capacity whereas, there is inverse relationship between IC₅₀ value and antioxidant activity. IC₅₀ value of ascorbic acid with DPPH assay in the present study was found to be 4.49±0.08 µg/ml which is similar to the IC₅₀ value of 5.94±0.28 µg/ml as reported by a previous study (Chludil et al., 2008). IC₅₀ values for TPA, TPHE, TPHM, TPE and TPM extracts were found to be 101.05±1.63, 48.51±1.00, 82.20±0.74, 176.24±4.08 and 114.45±1.85 µg/ml, respectively. Lowest IC₅₀ value was obtained for TPHE whereas highest was for TPE extract indicating that hydroethanolic extract was having best antioxidative activity whereas ethanolic extract proved the least. A previous study (Badmanaban et al., 2010) reported similar IC₅₀ values of 97.89 and 166.67 µg/ml for methanolic and aqueous extracts, respectively of T. portulacastrum. Another study (Yaqoob et al., 2014) reported the antioxidant activities of T. portulacastrum hydrolysates and found maximum activity in shoot followed by root and leaves which supports the selection of aerial (shoot) parts of the plant in our study.

Based on the findings of the phytochemical analyses and in vitro antioxidative studies, it can be clearly established that Trianthema portulacastrum has rich amounts of potent polyphenolic and flavonoid compounds having antioxidant properties. The presence of important polyphenols and flavonoids like rutin, gallic acid, protocatechuic acid, quercetin and myricetin in different extracts further affirms strong antioxidant potential. Hence, there is need to sub-fractionate the hydroethanolic extract of the plant for identification of novel compounds having distinct pharmacological properties.

We would like to thank G.B. Pant University of Agriculture and Technology, Pantnagar for providing facilities and support throughout our research. Facilities provided by AIRF, JNU, New Delhi are acknowledged.

The authors declare that they have no conflict of interests.