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. 2017 Jul 17;54(10):3082–3091. doi: 10.1007/s13197-017-2744-z

Extraction and identification of bioactive components in Sida cordata (Burm.f.) using gas chromatography–mass spectrometry

Mani Ganesh 1, Murugan Mohankumar 2,✉
PMCID: PMC5602971  PMID: 28974793

Abstract

Sida cordata (Burm.f.) is a pineal tropical plant in the family Malvaceae that is found throughout India and used to treat various diseases and ailments in many complementary and alternative medicine systems. This study identified the bioactive components present in whole-plant ethanol extracts of S. cordata using gas chromatography–mass spectrometry (GC–MS). Based on their retention times (RT) and mass-to-charge ratios (m/z), 29 bioactive compounds were identified: nonanoic acid, vitamin D3, 3-trifluroacetoxypentadecane, α-d-glucopyranoside, O-α-d-glucopyranosyl-(1.fwdarw.3)-α-d-fructofuranosyl,3,7,11,15-tetramethyl-2-hexadecan-1-ol, octadecanoic acid, ethyl ester, phytol, 9,12-octadecadienoic acid, methyl ester (E,E), 9,12,15-octadecadienoic acid, methyl ester (Z,Z,Z), oleic acid, 1,2-15,16-diepoxyhexadecane, 3-hexadecyloxycarbonyl-5-(2-hydroxyethyl)-4-methylimidazolium ion, methoxyacetic acid, 4-tetradecyl ester, 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester, 1-iodo-2-methylundecane, dodecane, 2,6,10-trimethyl-, 2-piperidinone-N-[4-bromo-n-butyl]-, squalene, octadecane-1-(ethenyloxy)-, Z,Z-2,5-pentadecadien-1-ol, 1-hexadecanol, 2-methyl-, spiro[androst-5ene-17,1′-cyclobutan]-2′-one-3-hydroxy-, (3a,17a)-, diethylene glycol monododecyl ether, vitamin E, cholestan-3-ol, 2-methylene-, (3a,5a)-, 2H-pyran, 2-(7-heptadecynyloxy)tetrahydro-, and cis-Z-α-bisabolene epoxide. The presence of various bioactive compounds justifies the use of this plant for treating various ailments by traditional practitioners.

Keywords: Sida cordata, GC–MS, Extraction, Bioactive compounds

Introduction

Aspects of the modern lifestyle, such as smoking, overconsumption of alcohol, and fast foods with excessive colorants and chemical preservatives place severe oxidative stress on cells and body systems, leading to the production of free radicals. These free radicals cause oxidative damage to lipids, proteins, and nucleic acids, which leads to diseases such as atherosclerosis, cancer, diabetes, inflammation, and Alzheimer’s and other degenerative diseases (Fransen et al. 2012). Many plant secondary metabolites are potential free radical scavengers, including flavonoids, anthocyanins, carotenoids, dietary glutathione, polyphenols, vitamins, and endogenous metabolites. Free radical scavengers are antioxidants that accept electrons from the free radicals produced in vivo or in vitro. Rutin, morin, quercetin (flavonoids), naringenin (flavone), catechin (flavanol), retinol and tocopherol (vitamins), and curcumin (polyphenol) are well-studied plant-derived secondary metabolites that possess anti-cancer, free radical scavenging, anti-ulcer, and antimicrobial activities. Flavanols are related to catechins, quercetin, and kaempferol, and their glycosides are found in beverages such as green and black teas and red wines. Quercetin occurs in onions and apples, while berries contain myricetin and quercetin. These dietary compounds protect against oxidative stress.

Many active pharmaceuticals have been derived from plant secondary metabolites, such as vinca alkaloids and Taxol, which effectively treat cancers (Cragg and Newman 2005). Hence, it is important to isolate natural antioxidants from plants. The initial steps are extraction and separation of the active phytochemicals from plants before identifying their active ingredients (Karimi and Jaafar 2011). Methods for identifying such compounds should be simple and repeatable. One of the best methods for identifying these compounds is gas chromatography–mass spectrometry (GC–MS), which can isolate and analyze compounds in a single step using a mass detector and available GC–MS libraries (Gomathi et al. 2015).

Sida cordata (Malvaceae) is a small perennial tropical weed found throughout India, as well as in some other Asian countries. It is commonly known as the long-stalk sida or kurunthotti in Tamil. In Indian alternative medicine, the entire plant is used for making medications such as Siddha and Ayurveda. Its roots are used as a diuretic to treat urinary problems and its seeds and oil extract are used as laxatives, aphrodisiacs, and demulcents. S. cordata is recommended in cystitis, colic gonorrhea, and piles (Gnanasekaran et al. 2012; Shah et al. 2014). The abortifacient effect of its ethanol extract has also been reported (Shah et al. 2014). It has hepatoprotective effects in vitro (Mistry et al. 2013; Shah et al. 2013). However, no study has identified the active constituents that are responsible for the therapeutic effects of S. cordata.

Therefore, we identified the active molecules present in this medicinally valuable plant using simple solvent extraction followed by GC–MS separation.

Materials and methods

Preparation of extract

Fresh whole S. cordata plants were collected from Tirupati, Andhra Pradesh, India. The plant material was identified taxonomically and authenticated by Professor. K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. A voucher specimen (no. 874) was stored in the department’s herbarium (Fig. 1). Approximately 5 kg of S. cordata were dried in the shade for 2 weeks, coarsely powdered, and sieved with #40 mesh. The dried material weighed about 1 kg. The powdered plant material was stored in an airtight container at room temperature until further use. Small quantities of the dried powdered material were preliminarily extracted with various solvents of different polarities, including ether, water, and ethanol. Ethanol was found to be the best solvent for extraction. Then, approximately 500 g of the dried powder was extracted with ethanol using hot continuous extraction with soxhlation at 60 °C. The extracts were then reduced to a dried powder using a rotary evaporator and stored at 4 °C until use.

Fig. 1.

Fig. 1

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Photographic Images of S. cordata a whole plant b magnified image of stem with flowering top

Qualitative phytochemical analysis

Preliminary evaluation of the extracted phytochemicals was performed using the methods of Harborne (1984), Trease and Evans (1989), and Kokate (1994) to test for the presence of steroids, carbohydrates, terpenoids, alkaloids, flavonoids, and saponins.

Gas chromatography–mass spectrometry analysis

The GC–MS analysis was performed using a Perkin-Elmer GC Clarus 500 gas chromatograph system interfaced with a mass spectrometer equipped with an Elite −5 MS column (5% diphenyl/95% dimethyl poly siloxane, 30 × 0.25 mm × 0.25 µm df). For GC–MS detection, an electron ionization system with ionizing energy of 70 eV and helium (99.999%) carrier gas at a constant flow rate of 1 mL/min, with an injection volume of 2 μL (extract dispersed in acetone, ultrasonicated for 15 min and filtered through a 0.22 µm nylon filter before injection), was injected with a split ratio of 10:1. The injector temperature was kept at 250 °C; the ion-source temperature was maintained at 200 °C. The oven temperature was programmed to increase from 110 °C (isothermal for 2 min) to 200 °C at 10 °C/min, and then to 280 °C at 5 °C/min; it was held at this temperature for 9 min. Mass spectra were then taken at 70 eV, with a scan interval of 0.5 s and fragments from 45 to 450 Da. The total GC run time was 36 min. The relative percentage of each component was calculated by comparing its average peak area to the total area using TurboMass software (ver. 5.2).

Compound identification

The isolated peaks of the plant extract were compared with known spectra in the National Institute of Standards and Technology database, which contains 62,000 patterns.

Results and discussion

Phytochemical profile

Preliminary investigation of the ethanol extract of S. cordata showed the presence of steroids, carbohydrates, terpenoids, alkaloids, flavonoids, and saponins (Table 1). Therefore, we selected the ethanol extract for further isolation studies using GC–MS.

Table 1.

Preliminary phytochemical screening results of ethanolic extract of Sida cordata

Phytoconstituents Test performed Ether Ethanolic extract Water
Alkaloids Mayer’s test − ++ +
Wager’s test − ++ +
Dragendorff test − ++ −
Hager’s test − ++ −
Terpenoids Liebermann test − ++ +
Salkowski test − ++ +
Saponins Forth test + + +
Foam test + + +
Carbohydrates Molisch’s test − + ++
Benedict’s test − + ++
Flavonoids Alkaline reagent test − ++ ++
Lead acetate test − ++ +
Glycosides Borntrager’s test + + +
Anthraquinones Sulphuric acid test − + −
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High concentration (++), Moderate concentration (+), Nil (−)

GC–MS analysis

Figure 2 shows a full scan gas chromatogram of the ethanol extract of S. cordata. It confirmed the presence of various bioactive compounds with different retention times (RT). The peaks of each component were obtained from the mass spectra and are shown in Fig. 3. The compounds identified by their RT, molecular weight, and percentage peak area are illustrated in Table 2, along with their molecular formulas. Table 3 summarizes the nature of the identified compounds and their biological activities, as predicted from Dr. Duke’s phytochemical and ethanobotanical databases (U.S. Department of Agriculture, Agricultural Research Service 1992–2016).

Fig. 2.

Fig. 2

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GC–MS traces of bio-active constituents of S. cordata ethanolic extract

Fig. 3.

Fig. 3

Fig. 3

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Mass spectrums of various phyto constituents indentified from the extract

Table 2.

Bio-active components identified in the S. cordata whole plant by GC–MS analysis

S. no. RT Name of the compound Mol. formula Mol. weight Peak area %
1 5.74 Nonanoic acid C9H18O2 158 14.18
2 6.91 Vitamin D3 C27H44O 384 23.53
3 8.44 3-Trifluroacetoxypentadecane C17H31F3O2 324 0.64
4 10.12 a-D-Glucopyranoside, O-a-D-glucopyranosyl-(1.fwdarw.3)-a-D-fructofuranosyl C18H32O16 504 5.61
5 11.07 3,7,11,15-Tetramethyl-2-hexadecan-1-ol C20H40O 296 3.74
6 12.81 Octadecanoic acid, ethyl ester C20H40O2 312 15.30
7 14.25 Phytol C20H40O 296 3.49
8 14.84 9,12-Octadecadienoic acid, methyl ester, (E,E)- C19H34O2 294 3.89
9 14.95 9,12,15-Octadecadienoic acid, methyl ester, (Z,Z,Z)- C19H32O2 292 23.89
10 15.26 Oleic acid C18H34O2 282 0.85
11 16.57 1,2-15,16-Diepoxyhexadecane C16H30O2 254 0.23
12 17.94 3-Hexadecyloxy carbonyl-5-(2-hydroxyethyl)-4-methylimidazolium ion C24H45N2O3 409 0.75
13 19.32 Methoxy acetic acid, 4-tetradecyl ester C17H34O3 286 1.55
14 19.99 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester C16H22O4 278 0.77
15 20.70 1-Iodo-2-methylundecane C12H25I 296 1.69
16 22.06 Dodecane, 2,6,10-trimethyl- C15H32 212 1.47
17 23.41 2-Piperidinone, N-[4-bromo-n-butyl]- C9H16BrNO 233 1.07
18 23.60 Squalene C30H50 410 1.69
19 24.74 Octadecane, 1-(ethenyloxy)- C20H40O 296 1.14
20 25.14 Z,Z-2,5-Pentadecadien-1-ol C15H28O 224 0.46
21 26.05 1-Hexadecanol, 2-methyl- C17H36O 256 0.50
22 26.91 Spiro[androst-5ene-17,1′-cyclobutan]-2′-one, 3-hydroxy-, (3a, 17a)- C22H32O2 328 0.84
23 27.33 Diethylene glycol monododecyl ether C16H34O3 274 0.84
24 27.84 Vitamin E C29H50O2 430 31.45
25 29.66 Cholestan-3-ol, 2-methylene-, (3a,5a)- C28H48O 400 1.31
26 30.72 2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro- C22H40O2 336 5.03
27 32.30 Cis-Z-a-Bisabolene epoxide C15H24O 220 1.37
28 32.89 9,12,15-Octadecatrienoic acid, 2-[(trimethylsilyl)oxy]-1-[[(trimethylsilyl)oxy]methyl]ethyl ester, (Z,Z,Z)- C27H52O4Si2 496 0.95
29 34.99 1-Heptatriacotanol C37H76O 536 1.80
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Table 3.

Activity of phytocomponents identified in the ethanolic extract of S.cordata whole plant

S. no. Name of the compound Nature Biological activity References
1 Nonanoic acid Carboxylic acid Antimicrobial Nurettin et al. 2006
2 Vitamin D3 Steroid Steroid hormone How et al. 1994
3 3-Tri fluro acetoxy pentadecane Acidic compound Anti-nephrotoxic and antioxidant activities Haider et al. 2016
4 a-D-Glucopyranoside, O-a-D-glucopyranosyl-(1.fwdarw.3)-a-D-fructofuranosyl Basic Sugars (Mono and Oligosaccharides) No activity reported –
5 3,7,11,15-Tetramethyl-2-hexadecen 1-ol Terpene alcohol Antimicrobial, anti-inflammatory Sudha et al, 2013
6 Octadecanoic acid, ethyl ester Stearic acid ester Antioxidant, anti-inflammatory Dr. Dukes
7 Phytol Diterpene alcohol Antinociceptive, Antioxidant, anticancer, anti-inflammatory, antimicrobial, diuretic, chemopreventive properties Camila et al. 2013
8 9,12-Octadecadienoic acid, methyl ester, (E,E)- Linolelaidic acid ester Hepatoprotective, antihistaminic, hypocholesterolemic, antieczemic Dr. Dukes
9 9,12,15-Octadecadienoic acid, methyl ester, (Z,Z,Z)- Linolenic acid, methyl ester Antiinflammatory, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Nematicide, Insectifuge, Antihistaminic, Antieczemic, Antiacne, 5-Alpha Reductase inhibitor, Antiandrogenic, Antiarthritic, Anticoronary, Insectifuge Rehana and Nagarajan 2013
10 Oleic acid Steric acid Anti-inflammatory, anti-androgenic, anti-cancer, preservative and hypocholesterolemic Sreekumar et al. 2014
11 1,2-15,16-diepoxyhexadecane Epoxide Antitumor, anti-inflammatory Imad et al. 2016
12 3-Hexadecyloxy carbonyl-5-(2-hydroxyethyl)-4-methylimidazolium ion Imidazole Antifungal, Antibacterial Subavathy and Thilaga 2016
13 Methoxyacetic acid, 4-tetradecyl ester Acidic compound Anti-microbial Agnel and Mohan 2014
14 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester Quinoline Cytotoxic Krishnan et al. 2014
15 1-Iodo-2-methylundecane Iodine compound Estrogen Achiraman et al. 2010
16 Dodecane, 2,6,10-trimethyl- Alkane No activity reported –
17 2-Piperidinone, N-[4-bromo-n-butyl]- Alkaloid Antimicrobial Anti-inflammatory Dr. Dukes
18 Squalene Triterpene Anti-oxidant, Anti-tumor Ryszard 2009
19 Octadecane, 1-(ethenyloxy)- Alkane No activity reported –
20 Z,Z-2,5-Pentadecadien-1-ol Unsaturated alcoholic compound No activity reported –
21 1-Hexadecanol, 2-methyl- Alcoholic compound Anti-microbial Sarada et al. 2011
22 Spiro[androst-5ene-17,1′-cyclobutan]-2′-one, 3-hydroxy-, (3a, 17a)- Steroid Antimicrobial, Anticancer, Anti-inflammatory, Diuretic, Anti-asthmatic, Anti-arthritic Archana et al. 2014
23 Diethylene glycol monododecyl ether Ether compound Surfactant Bandyopadhyay and Chanda 2003
24 Vitamin E Tocopherol Antioxidant Traber and Atkinson 2007
25 Cholestan-3-ol, 2-methylene-, (3a,5a)- Steroid Antimicrobial, anticancer, diuretic, anti-asthma, anti-arthritic Jegadeeswari et al. 2012
26 2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro- Flavonoid Antimicrobial Anti-inflammatory Antioxidant Amutha and Kottai 2014
27 Cis-Z-a-Bisabolene epoxide Pheromone compound To increase sex hormone activity Amutha and Kottai 2014
28 9,12,15-Octadecatrienoic acid, 2-[(trimethylsilyl)oxy]-1-[[(trimethylsilyl)oxy]methyl]ethyl ester, (Z,Z,Z)- Silica compound No activity reported –
29 1-Heptatriacotanol Alcoholic compound Anti-microbial Kalairasan et al. 2011
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Twenty-nine compounds were detected in the ethanol extract of S. cordata. Based on the RT and peak area of individual bioactive compounds, the predominant compounds were vitamin E (31.45%), 9,12,15-octadecadienoic acid, methyl ester, (Z,Z,Z) (23.89%), vitamin D3 (23.53%), octadecanoic acid, ethyl ester (15.30%), α-d-glucopyranoside, O-α-d-glucopyranosyl-(1.fwdarw.3)-α-d-fructofuranosyl (5.61%), 2H-pyran, 2-(7-heptadecynyloxy)tetrahydro- (5.03%),9,12-octadecadienoic acid, methyl ester, (E,E) (3.89%), 3,7,11,15-tetramethyl-2-hexadecan-1-ol (3.74%), and phytol (3.49%). Their chemical structures were predicted using the mass spectra based on their fragmentation, which generates peaks with different mass-to-charge ratios (m/z).

The use of medicinal plants in the treatment of various human ailments depends on their phytochemical constituents. This study revealed that the ethanol extract of S. cordata contained 29 compounds. Our preliminary investigation of the presence of various active constituents in water, ethanol, ether, and ethyl acetate extracts indicated that ethanol extracted the most phytochemicals from the plant. Hence, we used only the ethanol extract for the GC–MS study. Of the isolated compounds, nonanoic acid (Nurettin et al. 2006), 3,7,11,15-tetramethyl-2-hexadecen 1-ol (Sudha et al. 2013), methoxyacetic acid, 4-tetradecyl ester (Agnel and Mohan 2014), 2-piperidinone, N-[4-bromo-n-butyl]-, (Dr. Duke’s), 1-hexadecanol, 2-methyl- (Sarada et al. 2011), spiro[androst-5ene-17,1′-cyclobutan]-2′-one, 3-hydroxy-, (3a, 17a)-, cholestan-3-ol, 2-methylene-, (3a,5a)-, 2H-pyran, 2-(7-heptadecynyloxy)tetrahydro- (Archana et al. 2014), and 1-heptatriacotanol (Kalairasan et al. 2011) possess antimicrobial activity. The antioxidants included 3-trifluroacetoxypentadecane, octadecanoic acid, ethyl ester, squalene, vitamin E, 2-(7-heptadecynyloxy) tetrahydro-, and 2H-pyran (Rao et al. 1998; Traber and Atkinson 2007; Ryszard 2009; Amutha and Kottai 2014; Haider et al. 2016). cis-Z-α-bisabolene epoxide is a pheromone that increases sex hormone activity (Amutha and Kottai 2014), while 1-iodo-2-methylundecane acts as an estrogen (Achiraman et al. 2010). Eight compounds had cytotoxic activity (Camila et al. 2013): 9,12,15-octadecadienoic acid, methyl ester, (Z,Z,Z)- (Rehana and Nagarajan 2013), oleic acid (Sreekumar et al. 2014), 1,2-15,16-diepoxyhexadecane (Imad et al. 2016), 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester (Krishnan et al. 2014), squalene (Ryszard 2009, Kala et al. 2011), spiro[androst-5ene-17,1′-cyclobutan]-2′-one, 3-hydroxy-, (3a, 17a)- (Archana et al. 2014), and cholestan-3-ol, 2-methylene-, (3a,5a)- (Jegadeeswari et al. 2012). The phytols promoting reactive oxygen species constitute a promising novel class of pharmaceuticals for the treatment of rheumatic arthritis and possibly other chronic inflammatory diseases (Ogunlesi et al. 2009). No activities have yet been reported for α-d-glucopyranoside, O-α-d-glucopyranosyl-(1.fwdarw-3)-α-d-fructofuranosyl, a basic sugar moiety, octadecane, 1-(ethenyloxy)-, Z,Z-2,5-pentadecadien-1-ol, an ethanol compound, or 9,12,15-octadecatrienoic acid, 2-[(trimethylsilyl)oxy]-1-[[(trimethylsilyl)oxy]methyl]ethyl ester (Z,Z,Z).

Several active compounds are unique to S. cordata, including [androst-5ene-17,1′-cyclobutan]-2′one, cis-Z-α-bisabolene, 1-iodo-2-methylundecane cyclobutan]-2′-one, 3-hydroxy-, and (3a, 17a)-1,2-15,16-diepoxyhexadecane and phytol. Of these [androst-5ene-17,1′-cyclobutan]-2′-one, cis-Z-α-bisabolene and 1-iodo-2-methylundecane cyclobutan]-2′-have reported aphrodisiac and abortifacient activities, respectively (Shah et al. 2013); 3-hydroxy-, (3a, 17a)-1,2-15,16-diepoxyhexadecane may be responsible for the anti-inflammatory, antioxidant, and anti-cancer activity reported by Shah et al. (2014). Another phytosterol, vitamin E, may be responsible for the antioxidant activity.

This investigation revealed that S. cordata is a potential source of various bioactive compounds, such as esters, alcohols (Sarada et al. 2011), steroids (Archana et al. 2014; How et al. 1994; Kalpanadevi et al. 2012; Bandyopadhyay and Chanda 2003), alkaloids (Dr. Duke’s), terpenes (Sudha et al. 2013), and sugars, which justifies the use of this species in traditional medicine. Further studies need to examine molecules that are present at high concentrations and have potential biological activity. In the future, we plan to isolate compounds from different parts of S. cordata and evaluate their pharmacological activities.

Conclusion

Twenty-nine compounds were identified from the ethanol extract of whole S. cordata plants using GC–MS analysis. The presence of various bioactive compounds justifies the use of the whole plant for treating various ailments by practitioners of traditional Indian medicine. Some of the bioactive secondary metabolites identified may become commercially important phytopharmaceuticals. However, further studies are needed to ascertain their biological and pharmacological activity.

Acknowledgments

Author contributions

Dr. Mohan Kumar collected the plants, performed the extraction and data collection, and wrote the initial manuscript. Dr. Mani Ganesh contributed to the data interpretation and discussion and edited the final article.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.

Contributor Information

Mani Ganesh, Email: chemgans@gmail.com.

Murugan Mohankumar, Email: mmkpy6@gmail.com.

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