View of HPTLC, FTIR AND GC-MS ANALYSIS OF ETHANOL EXTRACT OF Talinum triangulare (Jacq.) Willd.

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 18009 - 18018 Received 05 March 2021; Accepted 01 April 2021.

18009 http://annalsofrscb.ro

HPTLC, FTIR AND GC-MS ANALYSIS OF ETHANOL EXTRACT OF Talinum triangulare (Jacq.) Willd.

1t. Ananthi*, ²k. Pravina

1,2 Pg And Research Department Of Biochemistry, S.T.E.T Women’s College (A), Sundarakkottai, Mannargudi – 614 016, Thiruvarur District, Tamil Nadu, India

*Corresponding author Email: [email protected]

ABSTRACT

Medicinal plants are the important bio resources of drugs for traditional system of medicine. They comprise of various natural dynamic fixings; in this manner they are utilized for the treatment of an extensive number of irresistible sicknesses. The aim of the present study to identify the phytoconstituents from the Talinum triangulare leaves using various techniques such as FTIR, HPTLC and GC-MS analysis.

Key words: FTIR, HPTLC and GC-MS analysis and Talinum triangulare

INTRODUCTION

India is the richest sources of medicinal plant which is used since time immemorial as the source of drugs for traditional systems of medicine. The plant extracts have been used by humans to protect him against several ailments and also to improve his health and lifestyle. Plants contain different phytochemicals like flavonoid, alkaloid, phenol and tannins, carboxylic acids, terpenes, amino acids and inorganic acids which has a specific distinctive medicinal property to plants (Mankilik et al., 2014).The biological properties in plants is analysed to determine the biological properties and there are a variety of techniques to determine and estimate the presence of such phytoconstituents in medicinal plants.

Fourier Transform Infrared Spectroscopy is a photochemical analytical technique that does not resolve that concentration of individual metabolites but provides a snapshot of the metabolic composition of a tissue at a given time. FT-IR can be employed to determine the structure unknown composition and the intensity of the chemical group. The FT-IR method determines the vibration of bonds within chemical functional group and generates a spectrum that can be regarded as a biochemical or metabolic “fingerprint” of the sample. By determining IR spectra from plant sample, it might possible to detect the minor changes of primary and secondary metabolites (Mccann et al., 1992). But on pharmacognosy, FT-IR is still a new tool to characterize and identify the commercial components from the adulterant and has been successfully utilized in the characterization of bacterial, fungal method and plant species (Helm et al., 1991).FT-IR is one of the most widely used methods to identify the chemical constituents and elucidate the compounds structure, and has been as a requisite method to identify medicines in pharmacopoeia of countries (Liu et al., 2006).

The phytochemicals of the plants are chemical compounds formed during the normal metabolic processes. These chemicals are often referred to as “Secondary metabolites”. The determination of phytoconstituents is largely performed by relatively expensive and often laborious techniques that are gas chromatography (GC) and liquid chromatography (LC) combined with specific detection schemes (Uzer et al., 2005; Eisenhauer et al.,2009). Analysis of chemicals has become easier and more cost-effective owing to the development of hyphenated chromatographic techniques, which are GC or LC-MS. GC-MS analysis can identify nature of compounds even less than 1mg present in the crude plant extract (Lieble et al.,1996). In recent years Gas Chromatography–Mass Spectrum (GC-MS), technique has been increasingly employed

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to analyze the secondary metabolites present in the medicinal plants, as this technique has been proved to be a best valuable method for the analysis of essential oil, alcohols, acids, esters, alkaloids, steroids, amino and nitro compounds (Muthulakshmi et al.,2012; Konovalona et al.,2013).

Modern high performance TLC (HPTLC) is an efficient instrumental analysis, and optimized quantitative HPTLC using a densitometric evaluation can produce result analogous to those obtained with gas chromatography (GC) and high performance liquid chromatography (HPTLC) (Wagner and Bladt, 2001).Thus, HPTLC ‘fingerprint analysis ‘may be a powerful tool for the quality control of raw plant material and may be an alternative technique, particularly in the analysis of crude plant extracts. An improvement over conventional TLC , HPTLC is an instrumental technique where by special plates and instrumental resources for sampling are used and the quantitative evaluation of separations is aided by densitometry (Nile and Park, 2014).

Talinum triangulare is one of vegetables widely cultivated and consumed in Africa especially Southern Nigeria (Burkill, 1994). The leaves of T. triangulare is eaten as a vegetable and employed in the treatment of several disease conditions such as measles, sexually transmitted diseases and internal heat and is associated with varying biochemical and physiological effects (Oluwole et al., 2003). The varying uses of the leaves of T. triangulare underscore its importance as major dietary supplement as a result of its nutritional and phytochemical composition (Ikeda et al., 2000).

MATERIALS AND METHODS

The leaves of Talinum traingulare were collected from in and around Mannargudi, Thiruvarur District. The collected plant materials were cleaned shade dried and coarsely powdered. The powdered plant material was used for further studies.

Preparation of Ethanol extract

100g plant powder was taken and subjected to Soxhlet extraction using 250 ml of the solvent ethanol. A solvent extracted fraction was dried in rotary vacuum evaporator to remove the traces of solvent and to obtain residues. The extract was stored at 4ºC in air tight continue for further investigation.

Preliminary Phytochemical Analysis

The plant extract was experimentally assessed for the existence of the phytochemical analysis by using the standard methods (Sofawara, 1993;Treas and Evans, 2002)

Fourier Transform Infrared (FT-IR) spectrum Analysis

The Plant samples were oven dried 60ºCand ground into fine powder through agate mortar.

Two milligrams of the sample were mixed with 200 mg KBr (FT-IR)and pressed into a pellet was immediately put into the sample holder and FT-IR spectra were recorded in the range 4000-450 CM‾¹ All the investigation were carried with a bruker 55 model FT- IR Spectrometer.

Gas chromatography Mass spectrum(GC-MS) Analysis

GC-MS analysis of the ethanol extract of T.triangulare was performed using a Perkin–

Elmer GC Clarus 500 system comprising an AOC-20i auto-sampler and a Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS) equipped with a Elite-5MS (5% diphenyl/95%

dimethyl poly siloxane) fused a capillary column (30 × 0.25 μm ID × 0.25 μm df). For GC-MS

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detection, an electron ionization system was operated in electron impact mode with an ionization energy of 70 eV. The mass-detector used in this analysis was Turbo-Mass Gold-Perkin-Elmer, and the software adopted to handle mass spectra and chromatograms was a Turbo-Mass ver-5.2.

High Performance Thin Layer Chromatography(HPTLC)

HPTLC take place in high speed capillary flow range of the mobile phase (Srivastava, 2011). Instrument used CAMAG TLC Scanner 3 "Scanner3-070408"S/N 070408(1.41.21) was used for detection and CAMAG Linomat 5 sample applicator was used for the application of the track. Double trough plate development chamber was used for development of chromatogram.

Software used was win CATS 1.4.3.

RESULTS AND DISCUSSION

Preliminary Phytochemical Screening

The results of qualitative analysis of ethanol extract of T.trianglare revealed the presence of alkaloids, flavonoids, tannins, phenols, saponin and absence of glycosides, carbohydrates and steroids (Table 1)

Table 1:Qualitative phytochemical screening of the leaf extract of T.triangulare

S.No. Phytoconstituents Result

1 Alkaloids +

2 Flavonoids +

3 Tannins +

4 Phenols +

5 Saponins +

6 Glycosides -

7 Carbohydrates -

8 Steroids -

+ indicates presence whereas – indicates absence

Alkaloid exhibited promising antidiarrheal, anti-inflammatory, anticancer andantidiabetic activities and cure urinary disorders (Awoyinka et al., 2007; Monika et al., 2012). Flavonoids are also known as a vitamin P elicit a wide range of therapeutic activities such as antihypertensive, antirheumatism, antidiuretic, antioxidant, antimicrobial and anticancer properties (Del et al.,1977;Veerachari et al.,2011). Tannin and phenols are acting as antimicrobial agents.

Many medicinally important secondary metabolites like alkaloids, flavonoids, steroids and triterpenoids were present in wild and tissue culture plant ethanol and methanol extracts.

Glycosides and Anthraquinones were present is wild and tissue culture plant ethanol extracts. This is many be due to the type and nature of the explants. These phytochemicals are known to have therapeutic importance since they have biological activities. Among the secondary metabolites, flavonoids have antibacterial activity (Cushnie and Lamb, 2005), phenols showed antioxidant activity (Chakraborthy et al., 2012), Tannins are shows to have antiviral, antitumor, wound healing and antiparasitic activities (Danial et al., 2011; Amokaha et al., 2002), Saponins are the precursors for the synthesis of steroidal drugs, sex hormones and contraceptives (Danial et al., 2011). The phytochemical study helps the future investigators regarding the selection of the particular explants for further study of isolating the active principles (Mishra et al., 2010).

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Fourier Transform Infra Red Spectra (FT - IR) Analysis

The FT-IR spectrum was used to identify the functional group of the active components based on the peak value in the region of infrared radiation. The ethanol extract of T.triangulare passed into the FT-IR and functional group of compounds were separated based on its peak ratio.

The ethanol extract of T.triangulare showed characteristic absorptions at 3418.79 Cm^-1 (for C=O stretch), 2922.84 Cm^-1(for C-H stretch), 2853.24 Cm^-1 (for C=O stretch), 1734.74 Cm^-1 (for C=O stretch), 1699.00 Cm^-1 (for N-H stretch), 1456.07 Cm^-1 (for C=O stretch), 1382.99 Cm^-1 (for C=O stretch), 1247.57 Cm^-1 (for C-H bending), 1158.78 Cm^-1 (for C-H stretch), 1041.65Cm^-1(for C=O stretch), 840.59 Cm^-1 (for C=C stretch), 722.22 Cm^-1 (for C-H stretch), 671.53Cm^-1(for C-H stretch), 605.70 Cm^-1 (for C-H stretch), and 469.86Cm‾¹ (for C-H stretch) respectively (Table 2 and Figure 1).

Table 2:FT-IR Analysis of ethanolic leaf extracts of T. triangulare

S.No

Group freqeuncy of sample

(Cm‾¹ )

Functional groups

Functional Assignment

1 3418. 79 C=O stretch Amines & Amides

2 2922.84 C=H stretch Aldehyde

3 2853.24 C=O stretch Carboxyl

4 1734.74 C=O stretch Carboxyl

5 1699.00 N-H stretch Amines

6 1456.07 C=O stretch Amides

7 1456.07 N=O stretch Nitro

8 1382.99 C=O stretch Carbonyl

9 1247.57 C-H bending Ketone

10 1158.78 C= H stretch Amine

11 1041.65 C=O stretch Polysaccharide

12 840.59 C=C stretch Aromatic

13 722.22 C-H stretch Halogen

14 671.53 C- H stretch Halogen

15 605.70 C-H stretch Iodo

16 469.86 C-H stretch Alkyl

The results were in accordance with the findings of Yamunadevi et al. (2012) also worked in FTIR analysis. The results of A. lanata root confirmed the presence of amines, amides, alkanes, aldehydes, ketones, esters, carboxylic acids, carbonyls, alkenes, primary amines, nitro compounds, aromatics, alcohols, esters, ethers and alkyl halides compounds

The characterization and antibacterial effect of plant-mediated silver nanoparticles using Ceropegiath waitesii was carried out by Muthukrishnan et al. (2015) and the presence of triterpenoids and methoxy groups played an important reduction role in the synthesis process was also authorized by them using FTIR. The absorbance bands analysis in bioreduction is observed in the region of 400–4000 cm1 are 1024.02, 1383.68, 1629.55, 2921.63 and 3449.30 Cm^-1. Major peaks were observed at 2921 cm1 that could be assigned to the C-H stretching vibrations of methyl, methylene and methoxy group (Muthukrishanan et al., 2015; Feng et al., 2009). But major peaks

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observed in the crude methanol extract of C. juncea are 3354.08 and 2838.40 that are indicating the presence of O-H alcohol and =C-H aldehyde groups.

The FTIR spectroscopic studies revealed the presences of alcohol, phenols, alkanes, alkynes, alkyl halides, aldehydes, carboxylic acid, aromatics, nitro compounds and amines were observed from ethanol leaf extract of Gmelina asiatica by Florence and Jeeva (2014).

Gas chromatography-Mass spectrum (GC-MS)Analysis

GC-MS is the best techniques to identify the constituents of volatile matter, long chain, branched chain hydrocarbons, alcohols acids, esters, etc. Peak area, retention time and molecular formula were used for the confirmation of phytochemical compounds. The compounds present in the ethanolic extract of T.triangulare, were identified by GC-MS analysis (Figure 2). The active principles with their retention time (RT), molecular formula, molecular weight (MW) and concentration (%) are presented in Table 3. Fifteen compounds were identified in ethanolic extract by GC-MS. The major constituents were identified in the ethanolic extract of T.triangulare were Stigmasta-5,22-DIEN-3-OL(3.BETA.,22E) (44.4), Campesterol (30.1), 1,54- dibromotetrapentacontane (5.89), Glycidyl oleate(3.67), Oleic acid (2.98), 9-Octadecenoic acid(E)- (2.78), Oxirane, hexadecyl-(1.74) and 9-Otadecenoic acid (Z)-, methyl Ester(1.27) and many other compounds were identified as low level. These phytochemicals are responsible for various pharmacological actions like antimicrobial and anti-oxidant anti-inflammation, Anti- cancer, Hepato protective, Diuretic, Antiasthma activities etc.

Table 3: GC-MS analysis of ethanolic leaf extract of T. triangulare

Peak Retention time

Area

%

Name of the Compound Molecular formula

Molecular Weight 1 6.352 0.32 ([(1Z)-1,3-Diphenyl-1-Pentenyl]Oxy )

(Trimethyl)Silane

C₂OH₂OSi 310

2 9.935 0.66 Cyclopentasiloxane, Decamethyl- C₁₀H₃₀O₅Si₅ 370 3 24.511 0.48 4-Hexen-1-Ol,2-Isopropenyl-5-

Methyl-, Acetate

C₁₂H₂₀O₂ 196

4 28.786 1.27 9-Octadecenoic Acid (Z)-, Methyl Ester

C₁₉H₃₆O₂ 296

5 28.882 0.89 Eicosenoic Acid, Methyl Ester C₂₁H₄₀O₂ 324

6 29.413 2.78 9-Octadecenoic acid, (E)- C₁₈H₃₄O₂ 282

7 29.484 2.98 Oleic Acid C₁₈H₃₄O₂ 282

8 33.457 1.5 Oleoyl chloride C₁₈H₃₃C10 300

9 33.538 0.84 Oleoyl chloride C₁₈H₃₃C10 300

10 34.101 3.67 Glycidyl oleate C₂₁H₃₈O₃ 338

11 34.184 2.47 Glycidyl oleate C₂₁H₃₈O₃ 338

12 38.381 30.1 Campesterol C₂₈H₄₈O 400

13 38.597 5.89 1,54-Dibromotetrapentacontane C₅₄H₁₀₈Br₂ 914 14 39.078 44.4 Stigmasta-5,22-Dien-3-Ol,

(3.Beta.,22e)-

C₂₉H₄₈O 412

15 39.541 1.74 Oxirane, hexadecyl- C₁₈H₃₆O 268

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Mass spectrometry becomes a vital tool in the hands of the organic chemists and biochemists because of its potential to supply the definitive, qualitative and quantitative information on molecules based on their structural compositions. Gas chromatography is attached to a Mass Spectrometer (GC-MS) enables mixture of small molecules mainly organic compounds of low molecular weight (<600) which can be analyzed.

Different crude extracts were obtained from the leaves of B. luzonica through selective sequential extraction with solvents of increasing polarity, namely, n-hexane, ethyl acetate and methanol. GC–MS analysis of the n-hexane, ethyl acetate and methanol extracts revealed the presence of various bioactive compounds. Phytol is present both in methanol (6.739%) and ethyl acetate extracts (20.288%) but in different quantities. In addition, squalene and 9,12,15- octadecatrienoic acid, methyl ester, (Z,Z,Z)- are both present in n-hexane and ethyl acetate extracts. The three crude extracts did not reveal a common major compound in them. Based on studies, some of the constituents revealed by GC–MS are biologically active compounds. They were proven to possess pharmacologic activities which may contribute to the healing potential of the plant. Phytol was proven to exhibit antioxidant and antinociceptive effects. Phytol, precursor of synthetic vitamin E and vitamin K, was found to be cytotoxic against breast cancer cell lines (MCF7) (Satyal et al.,2012). Propanetriol monoacetate was confirmed to be present in other plants exhibiting antimicrobial, anti-inflammatory, diuretic and anticancer effects (Foo et al., 2009). Propanetriol monoacetate was proven to be a precursor of tricetin (antifungal), but may also serve as a prodrug and vehicle for anticancer agents (Juneiouset al.,2014).

High Performance Thin Layer Chromatography (HPTLC) Analysis

The leaves extract of T.triangulare were analyzed for their fingerprinting by HPTLC method. The results from HPTLC fingerprint scanned at different Wavelength (254nm and 366nm) confirmed the presence of flavonid (Quercetin) at different concentrations (10 & 20µl). The photo of plate at 254nm showed the presence of 3 spots while at 366nm showed the presence 6 spots. The 3D display of the fingerprint profile and the peak display of ethanolic extract plant at 254nm and 366nm are presented in Figure 3-7. The display at 254nm shows the presence of 5 peaks. The Rf values of the peaks along with the area under the curve for

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each peak at 254 and 366 nm are tabulated in Table 4. Rf value of 0.74 observed in analysis confirmed the presence of flavonoid (Quercetin) in T.triangularae. Quantification indicated that the amount of Quercetin in ethanolic extract of Talinum triangularae was found to be 0.6016%w/w.

Table 4: Rf Values in HPTLC Fingerprint of extract of T. triangulare

Peak Start Rf

Start Height

Max Rf

Max Height

Height

%

End Rf

End Height

Area Area

%

Assigned Substance Standard

1 0.67 16 0.74 343.4 100.00 0.77 0.9 10259.2 100.00 Quercetin Plant Sample (10µl)

1 0.09 10.8 0.13 149.6 31.19 0.17 1.0 3750.5 27.14 unknown 2 0.54 10.2 0.60 33.6 7.16 0.62 24.0 1343.4 9.72 unknown 3 0.63 24.2 0.67 74.1 15.8 0.69 71.5 2155.1 15.60 unknown 4 0.69 71.6 0.72 211.6 45.13 0.81 0.9 6569.5 47.54 Quercetin Plant Sample (20µl)

1 0.03 0.1 0.06 12.1 1.72 0.07 11.1 185.2 0.79 unknown 2 0.07 11.2 0.13 195.2 27.71 0.17 0.0 5934.3 25.33 unknown

3 0.37 5.9 0.40 13.7 1.95 0.42 5.9 376.0 1.61 unknown

4 0.46 14.3 0.49 23.2 3.30 0.52 18.1 834.1 3.56 unknown 5 0.53 17.4 0.59 61.6 8.75 0.63 39.6 2831.9 15.09 unknown 6 0.63 40.1 0.67 114.9 16.31 0.68 106.4 3380.8 14.43 unknown 7 0.68 106.7 0.73 287.7 40.27 0.82 0.2 9881.4 42.19 Quercetin

HPTLC is the higher version of TLC and HPLC. It involves the similar approach and principle of separation of TLC i.e. adsorption. The modern qualitative and quantitative analysis version of traditional thin layer chromatography (TLC); High performance thin layer chromatography (HPTLC) is very popular and useful for its simplicity, authenticity and reliability.

WHO describes that there are three types of herbal medicines such as crude plant products, refined plant materials and herbal medicinal products (Gomathi et al., 2012). Due to the complex chemical structure of herbal products, HPTLC is used to analyze quantification of herbal products and active ingredients, phytochemical and biomedical products, check adulteration in herbal formulations (Goswami and Singh, 2019). The present investigation stated the presence of quercetin in the ethanolic extract of Talinum triangularae with the help of HPTLC analysis. Quercetin [2-(3, 4- dihydroxyphenyl)-3, 5, 7- trihydroxychromen-4-one] is a polyphenolic bioflavonoids contains flavonol mostly found in grapes, berries, onions, cherries, broccoli, citrus fruits (Choudhary and Sekhon, 2011). It is a plant pigment with versatile antioxidant potential along with anti- inflammatory, anti-allergic, antidiabetic, antihypertensive, free radical scavenging, vasodilator and chemo preventive properties (Shivatare et al., 2013). The similar method is used in the estimation of quercetin in the ethanolic extract of Catharanthus roseus. Linn leaves (Muhammet, 2016), Fenugreek Seeds (Trigonella foenum-graceum) and Azadirachta indica.

CONCLUSION

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The results showed that Talinum triangulare is endowed with a lot of bioactive constituents with various known pharmacological activities. Further research is needed to isolate bioactive constituents and may lead to the development of new drugs.

Acknowledgement

The authors are grateful to the Management of Sengamala Thayaar Educational Trust Womens’ College (A), Sundarakkottai, Mannargudi for their encouragement and support.

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