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Exploring the therapeutic potential of Cynanchum tunicatum (Retz.) Alston- assessment of phytochemicals and biological activities
⁎Corresponding author. dr.amuthaswaminathan@gmail.com (Amutha Swaminathan)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Cynanchum tunicatum (Retz.) Alston is native to the Asian region and is distributed in the tropical areas of India and Sri Lanka. The aim of this study is to explore the phytochemical composition, antioxidant potential, and anti-inflammatory properties of C. tunicatum. Metabolic profiling was carried out using phytochemical screening to detect and quantify the secondary metabolites. To evaluate the potential secondary metabolites using the standard methods, Fourier transform infrared (FTIR), High-Performance Thin Layer Chromatography (HPTLC) and Gas Chromatography–Mass Spectrometry (GC–MS) from organic extracts of C. tunicatum and its biological activities. FTIR investigated peaks that represent alkane and aromatic compounds. GC–MS revealed the presence of 22 constituents such as 1-Hexacosene (0.145 %), l-(+)-Ascorbic acid 2,6-dihexadecanoate (8.129 %), Campesterol (5.243 %), Beta −Amyrin (10.614 %), Lupeol (13.061 %), Octadecanoic acid (0.751 %) are the major active compounds present. HPTLC fingerprinting confirms the bioactive compounds such as colchicine, strychnine, coumarin etc. which are represented with corresponding Rf values. Among all the extracts of C. tunicatum methanolic extract showed highest antioxidant activities. In 2,2-diphenylpicrylhydrazyl method exhibit the IC50 of (38.91 µg/mL), Ferric reducing antioxidant power assay (1.6 µg/mL), total antioxidant assay (IC50 = 32.91 µg/mL) and IC50 of anti-inflammatory activity (42.31 µg/mL) respectively. These findings enrich the knowledge of the species Cynanchum tunicatum for the possible application as a source of bioactive compounds in drug discovery.
Keywords
Cynanchum tunicatum
Antioxidant activity
Anti-inflammatory activity
And phytochemical analysis
1 Introduction
Medicinal plants are employed in the treatment of a wide array of diseases and serve as abundant sources of secondary metabolites. Folklore medicine has been used from ancient period in several countries (Sridharan et al., 2022). In rural communities, traditional medicine practitioners were used for many diseases (Archana & Bose, 2022). The secondary metabolites significantly shapes the biological and pharmacological efficacy of the product, consequently posing substantial challenges to achieving reproducibility in preclinical investigations and clinical trials (Alcazar Magana et al., 2020).
Cynanchum tunicatum belongs to the family Apocynaceae, which comprises approximately 300 species, including some types of swallowworts. Most of these species are climbers or twiners. In Chinese medicine, different varieties of Cynanchum species has their essential phytochemical constituents are prescribed to treat fever, cough, pneumonia and asthma (Bailly et al., 2023). Recent pharmacological studies have demonstrated that Cynanchum plants possess significant pharmacological effects, such as anti-oxidation, immune regulation, anti-inflammatory, and anti-tumor. A literature survey shows that approximately 450 compounds have been isolated from various Cynanchum species (Han et al., 2018). Cynanchum species harbor a myriad of therapeutic compounds; however, so far, researchers have not explored the biological activity of the specific plant species C. tunicatum.
A number of compounds such as β-sitosterol, conduritol F, geniposide, wilfoside was identified in C. wilfordii and C. auriculatum. Literature survey evidences that the most important lacuna of bioactive compounds present in the C. tunicatum. The primary objective of this study is to explore the bioactive compounds and their biological properties. Fingerprinting of C. tunicatum was conducted using Gas Chromatography-Mass Spectrometry (GC–MS) and Fourier Transform Infrared (FTIR) spectroscopy. Additionally, High-Performance Thin Layer Chromatography (HPTLC) was employed as an effective technique for metabolite profiling.
2 Materials and methods
2.1 Plant material
The plant C. tunicatum was collected from Sirumalai forest, Dindigul, Tamil Nadu (N:10°16′45.1; E: 77 °59′55.1). The plant is maintaining in Herbal Garden, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore. The Plant was authenticated by Botanical Survey of India, Southern Region, Coimbatore (BSI/SRC/5/23/2023/Tech-551).
2.2 Solvents, chemicals, and extraction
Hexane, chloroform, ethyl acetate, methanol, toluene, acetone, formic acid, Mayer’s reagent Wagner’s reagent, Drangendorff’s reagent, Molisch’s reagent, Fehling reagent, Barfoed reagent, Bendict’s reagent, Borntrager’s reagent, sulphuric acid, ammonium solution, copper sulphate, sodium nitroprusside, ninhydrin, gallic acid, atropine, rutin, gallic acid and linalool, diclofenac sodium, DPPH reagent, Ascorbic acid, FRAP reagent, sulfuric acid, sodium phosphate, ammonium molybdate, Bovine serum albumin were used for further analysis. Atropine, Rutin, Ascorbic acid, Linalool from Sigma-Aldrich and other reagents were purchased from Sisco Research Laboratories.
Fresh and healthy plants were rinsed, shade-dried, and then pulverized. The extracts were obtained through sequential extraction of phytochemicals, transitioning from non-polar to polar solvents, employing hexane, chloroform, ethyl acetate, and methanol. All the crude extracts were dried using rotary vacuum evaporator (Roteva) and dissolved with respective solvents at mg/mL concentrations for further analysis.
2.3 Qualitative and quantitative phytochemical screening
The preliminary qualitative phytochemicals were analysed in all four extracts of C. tunicatum to determine the secondary metabolites (Harborne, 1998; Raaman, 2006). The Quantitative analysis of Alkaloids, Flavonoids, Phenols and Terpenoids were estimated by the following methods.
2.3.1 Total alkaloid content of C. tunicatum
The estimation of alkaloids was determined by bromocresol green method (Ajanal et al., 2012) for all four extracts of C. tunicatum. The extracts were dissolved in 2 N HCl (pH 2) and subsequently filtered. The resulting 10 mL solution was transferred to a separatory funnel and subjected to three washes with 50 mL of chloroform each. Following this, 5 mL of bromocresol green solution and 5 mL of phosphate buffer were added. Shake the mixture and extract yellow-coloured complex with 1-, 2-, 3- and 4-mL of chloroform in a separating funnel and collect the extract in 10 mL volumetric flask make up with chloroform. Atropine is used as a standard with different concentrations (0.2, 0.4, 0.6, 0.8 and 1 µg/ mL). The absorbance was measured using a spectrophotometer at 470 nm.
2.3.2 Total flavonoid content of C. tunicatum
The aluminium chloride method (Slinkard & Singleton, 1977) was used for flavonoid estimation using various extracts of C. tunicatum. Take 0.5 mL of all four extracts and mixed with 0.1 mL of 10 % aluminium chloride, 0.1 mL of potassium acetate and 2.8 mL of distilled water. It incubates at room temperature for 30 min. Rutin was used as a standard with various concentrations (0.2, 0.4, 0.6, 0.8, 1 µg/mL). The absorbance of the reaction mixture was measured at 415 nm.
2.3.3 Total Phenol content of C. tunicatum
Total phenolics were determined by Folin Ciocalteau (Slinkard & Singleton, 1977), using various extracts of C. tunicatum. One mL of extract was added with 1.0 mL of Folin Ciocalteau reagent followed by the addition of 3.0 mL of 2 % sodium carbonate and the mixture kept for 2 hrs incubation in dark condition. Gallic acid used as a standard. The blue colour indicates the presence of phenols and the absorbance was measured at 760 nm.
2.3.4 Determination of total terpenoid content of Cynanchum tunicatum
The total terpenoid content was determined by colorimetry assay (Indumathi et al., 2014) using various extracts of C. tunicatum. One mL of plant extract was mixed with 150 µL of vanillin-glacial acetic acid solution (5 %) followed by the addition of 500 µL of perchloric acid. The mixture was kept under water bath for 45 mins at 60℃. Glacial acetic acid (2.25 mL) was added to the mixture and observed reddish pink to blue color. The absorbance was measured at 548 nm. Linalool is used as a standard.
2.4 Determination of antioxidant activity
2.4.1 DPPH assay of C. tunicatum
The DPPH free radical scavenging activity was assessed according to the method described by Hatano et al. (1988). All four extracts of C. tunicatum at various concentrations (20, 40, 60, 80, 100 µg/mL) were combined with 2.7 mL of a 0.1 mM methanolic solution of DPPH. The resulting mixture was vigorously shaken and allowed to incubate in the dark for 30 min at room temperature. Subsequently, the absorbance was measured at 517 nm. A control sample, devoid of any extract but containing DPPH, was also measured. The percentage inhibition and IC50 value were then calculated. The absorbance of positive control is a pure compound. Hence it is showed higher inhibition percentage than plant crude extracts.
2.4.2 FRAP − assay of C. tunicatum
The capacity to reduce ferric ions was evaluated using a modified protocol based on the method outlined by Benzie & Strain, (1996). A portion of each extract, spanning various concentrations (20, 40, 60, 80, 100 µg/mL), was combined with 3 mL of FRAP reagent composed of 10 parts of 300 mM sodium acetate buffer (pH 3.6), 1 part of 10 mM 2,4,6-Tripyridyl-S-Triazine solution, and 1 part of 20 mM FeCl3 6H2O solution. The reaction mixture was then incubated in a water bath at 37℃ for 30 min. Gallic acid served as the standard reference. Subsequently, the absorbance was recorded at 593 nm, and the FRAP value was computed.
2.4.3 Total antioxidant activity of C. tunicatum
The total antioxidant capacity of C. tunicatum was assessed using the phosphomolybdenum method as described by (Prieto et al., 1999). A 0.5 mL aliquot of each of the four extracts, prepared at various concentrations (20, 40, 60, 80, 100 µg/mL), was combined with 4.5 mL of phosphomolybdenum reagent, consisting of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The mixtures were then incubated in a water bath at 95℃ for 90 min. Ascorbic acid served as the standard reference. Subsequently, the absorbance was measured at 695 nm, and the inhibition percentage and IC50 value were determined.
2.5 Evaluation of anti-inflammatory activity
The anti-inflammatory activity of all four extracts of C. tunicatum was assessed using the bovine serum albumin denaturation assay, as described by Chandra et al., (2012). A reaction mixture of 5 mL was prepared, comprising 0.2 mL of bovine serum albumin, 2.8 mL of phosphate-buffered saline (pH 6.4), and 2 mL of various concentrations (20, 40, 60, 80, 100 µg/mL) of plant extracts. The mixtures were then incubated at (37 ± 2) ℃ in a Biochemical Oxygen Demand (BOD) incubator for 15 min, followed by heating at 70℃ for 5 min. Subsequently, the absorbance was measured at 660 nm, with diclofenac sodium serving as the standard reference. Percentage inhibition (%) = [Sample absorbance/(Control absorbance − 1)] × 100
2.6 FTIR (Fourier transform infrared) analysis
FTIR (Shimadzu Miracle 10) equipped with a temperature-stabilized detector was used for the analysis of functional groups present in phytochemicals in the range of 400–4000 cm−1 with a resolution of 16 cm−1. The characteristic peaks and their functional groups were detected and recorded for further structure elucidated using FTIR.
2.7 GC–MS analysis
Mass spectrometry data were utilized in conjunction with GC–MS/MS analysis to identify compounds present within the extract. Interpretation of mass spectra was facilitated using the comprehensive database provided by the National Institute of Standards and Technology (NIST), which encompasses over 62,000 patterns. Unknown components were matched against known compounds stored within the NIST library. The identification process involved determination of the name, molecular weight, and molecular formula of the extract samples (Valdez et al., 2018).
2.8 HPTLC fingerprinting analysis
HPTLC profiling is used for the separation, identification, determination, and validation of phytoconstituents such as alkaloids, flavonoids, phenols and terpenoids from methanolic extracts of C. tunicatum. Aliquot of the test solution (100 mg / mL) and the standard solution were loaded as 5 mm band length in a silica gel 60F254TLC plate using a Hamilton syringe and LINOMAT 5 instrument (CAMAG, Muttenz, Switzerland). The samples were loaded in TLC plate with respective mobile phases and the images were captured in white light and UV light at 254 nm and 366 nm of developed and derivatized plates.
2.9 Statistical analysis
The experimental results were expressed as mean ± standard deviation. Statistical significance was evaluated by one-way analysis of variance (ANOVA). Values of p ≤ 0.05 were significant using Tukey’s test.
3 Results and discussion
3.1 Phytochemical analysis
The preliminary qualitative analysis was performed in four different extracts of C. tunicatum. The secondary metabolites are rich in ethyl acetate and methanolic extracts such as alkaloids, carbohydrates and glycosides, phenol and terpenoids, quinine, and phytosteroids (Table 1). Similar results were obtained in genus Cynanchum such as steroids, saponins, alkaloids, flavonoids, phenols and terpenoids (Han et al., 2018). According to Shrivastava et al., analyzed the terpenoids, flavonoids, tannins, saponins, steroids, carbohydrates, and alkaloids in methanolic fruit extract of Trichosanthes dioica (Shrivastava et al., 2021). In Ricinus communis, Rahman et al. studied the plant secondary metabolites in seed extract such as terpenoids, flavonoids, coumarin, steroids, and reducing sugar. These secondary metabolites are exclusively significant for the plant defence mechanism (Rahman et al., 2022). −: Absent, +: Present, ++: Strongly Present.
S.No
Tests
Hexane
Chloroform
Ethyl acetate
Methanol
1
A
Alkaloids
Mayer’s Test
−
−
++
++
B
Wagner’s Test
++
−
+
+
C
Drangendorff’s Test
+
−
−
+
2
Flavonoids
+
−
++
++
3
A
Carbohydrates and glycosides
Molisch’s Test
++
++
+
+
B
Fehling Test
−
+
++
+
C
Barfoed Test
+
+
++
++
D
Bendict’s Test
−
−
++
++
E
Borntrager’s Test
+
+
+
++
4
Saponin Test
−
−
+
+
5
A
Oils and fats test
Spot Test
−
−
−
+
6
A
Phenolic and terpenoid test
Ferric chloride Test
−
−
+
++
B
Gelatin Test
−
−
−
++
C
Lead Acetate Test
++
−
−
++
D
Alkaline Reagent Test
−
−
++
++
E
Magnesium and Hydrochloric Acid Test
+
−
−
++
F
Phlobaterpenoids
−
−
−
−
7
Quinone
++
−
++
+
8
Glycosides
++
−
++
++
9
Cardiac glycosides
+
−
−
−
10
Terpenoid
++
−
+
+
11
Coumarins
−
−
−
+
12
Steroids
−
−
+
+
13
Phytosterols
−
++
++
++
14
Protein
+
−
++
+
3.1.1 Estimation of total alkaloid content of Cynanchum tunicatum
Alkaloids are specific remedy for many diseases particularly in mammals because of their general toxicity, deterrence capability, adaptogenic activities and anti-inflammatory activity which help to alleviate pain endurance against stress and resistance to diseases (Zhang & Hu, 2020). In C. tunicatum the alkaloid contents were quantified in all the extract. Interestingly, the maximum alkaloid content was found in methanolic extract (1.37 ± 0.03 µg/mL) followed by ethyl acetate extract (0.78 ± 0.05 µg/mL), hexane (0.67 ± 0.03 µg/mL), chloroform (0.53 ± 0.04 µg/mL) of C. tunicatum (Fig. 1(e)). Atropine is used as a reference standard (Fig. 1(a)). Nagalakshmi et al. also studied that the maximum alkaloid content (5.62 mg/g) in methanolic extract of Tinospora cordifolia was observed (Nagalakshmi et al., 2023).
a) Calibration curve of standard atropine for the quantification of total alkaloid content b) Calibration curve of standard rutin for the quantification of total flavonoid content c) Calibration curve of standard gallic acid for the quantification of total phenol content d) Calibration curve of standard linalool for the quantification of total terpenoid content e) Graphical representation of quantitative secondary metabolite screening of Cynanchum tunicatum.
3.1.2 Total flavonoid content of C. tunicatum
The total flavonoid content was determined using aluminium chloride method of various solvents. The yellow colour represented the presence of flavonoid. The maximum flavonoid content was observed in methanolic extracts (4.23 ± 0.05 µg/mL) followed by ethyl acetate (2.37 ± 0.01 µg/mL), chloroform (1.28 ± 0.02 µg/mL) and hexane (0.104 ± 0.01 µg/mL) (Fig. 1(e)). Rutin is used for a reference standard (Fig. 1(b)). Interestingly, Garg & Garg, analysed flavonoid content in methanolic leaf extract of Ocimum sanctum, obtained the maximum amount (4.75 mg /100 mg) and it is correlated with antioxidant activity (Garg & Garg, 2019).
3.1.3 Total Phenol content of C. tunicatum
The Phenolic content of hexane, chloroform, ethyl acetate and methanolic extracts of C. tunicatum showed 1.33 ± 0.01 µg/mL, 1.35 ± 0.007 µg/mL, 2.27 ± 0.063 µg/mL and 2.32 ± 0.037 µg/mL respectively, in which methanolic extract showed the maximum amounts of phenols (Fig. 1 (e)). Gallic acid is used as a reference standard (Fig. 1(c)). Similarly, the previous researchers also analyzed the flavonoid content in various medicinal plants. Arya et al. studied the methanolic leaf extract of Cichorium intybus containing maximum phenolic content (302 ± 0.251 mg/mL) (Arya et al., 2022). Ahmed et al. investigated the methanolic extract of Cannabis sativa encompassing the maximum phenolic content (36.42 ± 1.905 µg/mL) (Abdel-Azeem et al., 2020).
3.1.4 Total terpenoid content of C. tunicatum
Terpenoid contents were analysed using the folin–ciocalteu method using various solvents of C. tunicatum. The maximum content of terpenoid was observed in methanol (1.60 ± 0.02 µg/mL) followed by ethyl acetate (1.57 ± 0.14 µg/mL), hexane (1.18 ± 0.02 µg/mL) and chloroform (0.86 ± 0.02 µg/mL) (Fig. 1 (e)). Linalool is used as a standard (Fig. 1 (d)). (Indumathi et al., 2014) analysed the highest terpenoid content in the methanolic leaf extract of Enicostemma litorrale. Terpenoids are significantly helpful for therapeutic properties like anticancer, antimicrobial and antioxidant activity (Roaa, 2020).
Preliminary phytochemical investigation of C. tunicatum facilitates the identification of phytochemical constituents and is quantified in the ranked as flavonoids > phenols > terpenoids > alkaloid. The differences in the values were statistically significant (p ≤ 0.05). Based on the previous literature (Wang et al., 2021), Cynanchum species contains 232 compounds including alkaloids, terpenoids, C21 steroids, flavonoids, acetophenones and these phytochemical compounds are abundantly present in C. tunicatum. In future, based on the secondary metabolites biological activities will be carried out to confirm the medicinal properties of the C. tunicatum.
3.2 Antioxidant assays
Reactive oxygen species (ROS) damage the cells through pollution, radiations, and other environmental factors. These damages were protected through antioxidants which are the molecules that can neutralize the ROS by donating the electrons and stabilizes hazardous free radicals. The mechanisms of antioxidant activity either directly or indirectly by inhibiting the free radical damage, thereby preventing the nucleic acids, proteins, lipids, and other molecules. Plant derived bioactive compounds protect the cells from free radical damage by the free radical scavenging activity. In this present study, bioactive compounds obtained from Cynanchum tunicatum extracts showed more efficient antioxidant potential was analysed by DPPH, FRAP, and TAA.
3.2.1 DPPH assay of C. tunicatum
Free radicals cause damage to the plant cells, whereas antioxidants play a crucial role in protecting the cells. Natural antioxidants such as phenols and flavonoids have the potential to provide resistance against free radical-induced oxidative stress (Karale et al., 2022). The antioxidant activity was anlayzed for all four crude extracts of C. tunicatum using DPPH assay. The maximum inhibition percentage was obtained in the methanolic extract (IC50 = 38.91 µg/mL) followed by ethyl acetate (IC50 = 52.26 µg/mL) (Table 2). Ascorbic acid was used as a reference standard and its IC50 value is 53.101 µg/mL. This result confirmed the methanolic extract of C. tunicatum have potential for antioxidant activity. Still there is no previous antioxidant study were found on C. tunicatum. Akgül et al. reported that Ethyl acetate extract showed maximum inhibition (68.721 ± 1.694) of free radical scavenging activity in Euphorbia eriophora (Akgül et al., 2022). All values were expressed in mean ± standard deviations of triplicate measures. Each analysis was statistically significant (p ≤ 0.05), ‘a’ expressed best results and ‘e’ showed poor results.
Concentration (µg/mL)
Percent scavenging of stable DPPH free radical (%)
Hexane
Chloroform
Ethyl acetate
Methanol
Ascorbic acid
20
8.67 ± 0.003 e
26.4 ± 0.01 e
29.3 ± 0.004 e
40.3 ± 0.007 e
22.76 ± 0.004 e
40
20.7 ± 0.003 d
36.4 ± 0.006 d
39.4 ± 0.009 d
52.09 ± 0.004 d
40.4 ± 0.004 d
60
35.01 ± 0.002c
48.4 ± 0.005c
55.6 ± 0.002c
57.9 ± 0.01c
56.05 ± 0.007c
80
54.1 ± 0.008b
58.3 ± 0.005b
68.1 ± 0.005b
72.5 ± 0.004b
71.39 ± 0.006b
100
69.1 ± 0.004a
70.8 ± 0.01 a
84.08 ± 0.01 a
85.9 ± 0.005 a
86.82 ± 0.002 a
IC50
76.15
63.405
52.26
38.91
53.101
3.2.2 FRAP assay of C. tunicatum
The determination of antioxidant activity, based on their ferric-reducing power, involves assessing the ability to reduce Fe3 + to Fe2+ (Ene-Obong et al., 2018). The antioxidant activity of all four crude extracts of C. tunicatum by using ferrous reducing assay. The maximum reduction was observed at a concentration of 100 µg/mL in methanolic extract (1.6 ± 0.11) followed by ethyl acetate (1.2 ± 0.007), chloroform (1.09 ± 0.009) and hexane (0.98 ± 0.007) (Table 3). The ferric-reducing assay of methanolic extract shows higher antioxidant capacity compared to gallic acid. According to Noreen et al. reported that the ethanolic extract of Coronopus didymus (aerial parts) observed the highest Optical Density (OD) value is 0.304 at the concentration of 50 µg/mL (Noreen et al., 2017). All values were expressed in mean ± standard deviations of triplicate measures. Each analysis was statistically significant (p ≤ 0.05), ‘a’ expressed best results and ‘e’ showed poor results.
Concentration (µg/mL)
FRAP value
Hexane
Chloroform
Ethyl acetate
Methanol
20
0.02 ± 0.0005 e
0.24 ± 0.009 e
0.11 ± 0.005 e
0.29 ± 0.01 e
40
0.16 ± 0.01 d
0.43 ± 0.01 d
0.40 ± 0.01 d
0.45 ± 0.009 d
60
0.44 ± 0.01c
0.59 ± 0.01c
0.63 ± 0.02c
0.89 ± 0.03c
80
0.65 ± 0.004b
0.84 ± 0.001b
1.008 ± 0.009b
1.20 ± 0.01b
100
0.98 ± 0.007 a
1.09 ± 0.009 a
1.20 ± 0.007 a
1.60 ± 0.11 a
3.2.3 Total antioxidant activity of C. tunicatum
The total antioxidant activity is based on the phosphomolybdate reagent which reduces Molybdenum VI to V with maximum absorption and it forms a green phosphate/Mo (V) complex. All four extracts of C. tunicatum with various concentrations were analysed and the maximum activity was observed at 100 µg/mL in methanol extract (IC50 = 32.91 µg/mL) followed by ethyl acetate (IC50 = 47.11 µg/mL), chloroform (IC50 = 86 µg/mL) and hexane (IC50 = 82.11 µg/mL) (Table 4). Hence the C. tunicatum is a potential candidate of antioxidant property. Ascorbic acid was used as a standard (IC50 = 53.38 µg/mL). In the previous research evidenced that the ethanolic leaf extract of Limonia acidissima showed maximum antioxidant activity of 5.055 µL (Parvez & Sarker, 2021). Bayliak et al., studied that highest reducing ability of Rhodiola rosea aqueous extract was 3.66 ± 0.24 mg/mL (Bayliak et al., 2016). All values were expressed in mean ± standard deviations of triplicate measures. Each analysis was statistically significant (p ≤ 0.05), ‘a’ expressed best results and ‘e’ showed poor results.
Concentration
(µg/mL)Percent scavenging of TAA (%)
Hexane
Chloroform
Ethyl acetate
Methanol
Ascorbic acid
20
13.6 ± 0.03 e
15.4 ± 0.07 e
29.9 ± 0.01 e
44.2 ± 0.005 e
27.3 ± 0.02 e
40
21.1 ± 0.023 d
17.5 ± 0.047 d
48.0 ± 0.008 d
54.1 ± 0.008 d
44 ± 0.02 d
60
30.0 ± 0.003c
31.2 ± 0.027c
60.3 ± 0.007c
62.4 ± 0.007c
53.1 ± 0.01c
80
57.0 ± 0.013b
45.1 ± 0.057b
70.3 ± 0.009b
75.1 ± 0.002b
68.1 ± 0.02b
100
58.6 ± 0.023 a
62.1 ± 0.017 a
81.8 ± 0.006 a
86.3 ± 0.003 a
83.6 ± 0.002 a
3.3 Evaluation of anti-inflammatory activity assay
Protein is denatured by harmful pathogens or free radicals and inhibiting this process can be a potential mechanism for anti-inflammatory activity (Yesmin et al., 2020). Result displayed in Table 5 show IC50 inhibition of albumin denaturation of all the extracts of C. tunicatum at concentration between 42.31 to 68.44 μg/mL in the following order of methanol > ethyl acetate > hexane > chloroform. The standard diclofenac sodium an anti-inflammatory drug showed IC50 value at 60.14 μg/mL. According to Phoenix dactylifera (Jihl − a variety of date seed) showed a high inhibition (IC50 = 90.34 μg/mL) in scavenging nitric oxide free radicals and possessed the highest anti-denaturation effect (Hmidani et al., 2020). Similarly, Saleem et al. analysed BSA assay in Moringa oleifera showed maximum inhibition percentage of protein denaturation with IC50 value of butanol > n-hexane > ethyl acetate > piroxicam > diclofenac sodium > methanolic > aqueous (Saleem et al., 2020). In this present study, the methanolic extract contains alkaloids, flavonoids, phenols and terpenoids are the main factors influenced the anti-inflammatory activity. All values were expressed in mean ± standard deviations of triplicate measures. Each analysis was statistically significant (p ≤ 0.05), ‘a’ expressed best results and ‘e’ showed poor results.
Concentration
(µg/mL)Percent inhibition of anti-inflammatory (%)
Hexane
Chloroform
Ethyl acetate
Methanol
Diclofenac sodium
20
24.8 ± 0.002e
2.51 ± 0.0005e
13.22 ± 0.0005e
49.2 ± 0.002e
39.3 ± 0.003e
40
118.009 ± 0.002d
29.5 ± 0.003d
28.1 ± 0.003d
71.01 ± 0.002d
122.09 ± 0.01d
60
194.6 ± 0.001c
62.8 ± 0.002c
50.7 ± 0.002c
102.8 ± 0.001c
221.7 ± 0.007c
80
268.08 ± 0.003b
97.4 ± 0.001b
71.4 ± 0.001b
147.8 ± 0.004b
301.1 ± 0.004b
100
310.7 ± 0.002 a
130.8 ± 0.002 a
85.7 ± 0.002a
171.01 ± 0.001a
374.5 ± 0.009a
IC50
59.23
68.44
57.03
42.31
60.14
3.4 FTIR analysis of C. tunicatum
FTIR spectrum of whole plant of C. tunicatum from hexane, chloroform, ethyl acetate and methanol crude extracts were analysed to identify the functional groups. The information in Fig. 2 showed peak values along with their functional group of various extracts, respectively. The main peak observed in FTIR spectra of hexane extract at 2870.08 cm−1 (medium), 2924.09 cm−1 (strong), 2954.95 cm−1 (narrow sharp strong), 1728.22 cm−1 (weak), 1465.90 cm−1 (strong), 1381.03 cm−1 (medium), 1242.16 cm−1 (weak), 1172.72 cm−1 (weak), 1041.56 cm−1 (weak), 887.26 cm−1 (medium), 725.23 cm−1 (strong), 817.82 cm−1 (weak), 563.21 cm−1 (weak). Infrared spectrum of chemical constituents of chloroform exhibited peaks at 3325.28 cm−1 (broad strong), 2947.23 cm−1 (medium), 2831.50 cm−1 (sharp medium), 1666.50 cm−1 (very weak), 1404.18 cm−1 (weak), 1026.13 cm−1 (strong) and 686.66 cm−1 (medium). The peak of ethyl acetate represented at 3633.89 cm−1 (weak), 2985.81 cm−1 (sharp medium), 2900.94 cm−1 (very weak), 1735.93 cm−1 (sharp narrow strong), 1442.75 cm−1 (medium), 1373.32 cm−1 (sharp medium), 1234.44 cm−1 (sharp strong), 1095.57 cm−1 (medium), 1041.56 cm−1 (sharp narrow strong), 786.96 cm−1 (broad medium), 848.68 cm−1 (sharp medium), 632.65 cm−1 (medium), 609.51 cm−1 (medium). The FTIR spectra of methanol extract showed peaks at 3317.56 cm−1 (broad medium), 2831.50 cm−1 (sharp medium), 2939.52 cm−1 (medium), 2522.89 cm−1 (weak), 2229.71 cm−1 (very weak), 2044.54 cm−1 (weak), 1666.50 cm−1 (weak), 1450.47 cm−1 (medium), 1111.00 cm−1 (medium), 1018.41 cm−1 (narrow strong), 671.23 cm−1 (broad medium), 601.79 cm−1 (medium), 524.64 cm−1 (medium).
Infrared spectra with Fourier transform in the fingerprint region of a) Hexane extract, b) Chloroform extract, c) Ethyl acetate extract and d) Methanol extract of Cynanchum tunicatum.
The spectra of C. tunicatum with the frequency range from 2831.50 to 2985.81 represented the presence of alkane group (C–H stretch). The peak at 3325.28 and 3633.89 corresponds to the presence of alcohol (O–H stretch) (Vahur et al., 2016). The peak at 2522.89 was observed as carboxylic acid (O–H stretch). The peak at 2229.71 showed the presence of nitriles (C≡N stretch). The peak at 2044.54 signified the presence of isothiocyanate (N = C = S stretch). The peak at 1666.50 and 1728.22 to 1735.93 unveiled the presence of aromatic compounds (C–H bending). The peak at 1450.47 and 1465.90 displayed alkane (O–H bending) (Reignier et al., 2021). The peak at 1404.18 and 1442.75 showed carboxylic acid (O–H bending). The peak at 1381.03 and 1373.32 exhibited alcohol (O–H bending). The peak ranges from 1172.72 to 1242.16 represented by amine (C–H stretch). The peak ranges from 1018.41 to 1041.56 showed a sulfoxide group (S = O stretch). The medium peak at 887.26 exhibited alkene (C = C bending). The peak ranges from 563.21 to 848.68 showed a halo compound (C-Cl stretch). The medium peak at 524.64 contained a halo compound (C-I stretch) (Papakosta et al., 2020). The FTIR spectrum conforms to the presence of alkaloids, flavonoids, phenols, terpenoids, carboxylic acid, alkane, aromatic alkenes in C. tunicatum.
3.5 GC–MS analysis
The GC–MS analyses of the methanolic extract of the whole plant of C. tunicatum provided the separation of 22 components, from 11 different groups were well identified using their mass spectra and retention index data. The major compounds are 1-Hexacosene (0.145 %), 17-(1,5-Dimethylhexyl)-10,13-dimethyl-2 (0.180 %), 1,3-Dioxolane, 4-methyl-2-pentadecyl (0.382 %), 7-Dehydrodiosgenin (0.155 %), Methyl Palmitoleate (0.119 %), Palmitic acid (0.988 %), Methyl (Z)-5,11,14,17-eicosatetraenoate (0.13 %), Octadecanoic acid (0.751 %), Myristic acid (0.141 %), Methyl linoleate (0.147 %), l-(+)-Ascorbic acid 2,6-dihexadecanoate (8.129 %), Campesterol (5.243 %), Phenol, 2,6-dimethoxy-4-(2-propenyl)- (0.108 %), 4.alpha.,14-Dimethyl-5. alpha-ergosta-8 (0.430 %), Pregn-5-en-20-one, 12-(acetyloxy)-3,8,14 (0.136 %), Cholesta-22,24-dien-5-ol, 4,4-dimethyl- (3.482 %), Lupeol (13.061 %), Megastigmatrienone (0.116 %), Lupenone (0.306 %), Beta −Amyrin (10.614 %), 17-(1,5-Dimethyl-3-phenylthiohex-4-enyl) (0.593 %) and Phytol (1.455 %). In Table 6 represents the compound name, molecular formula, retention time, peak area, functional group and reported medicinal applications for methanol extract of C. tunicatum. The structure of these metabolites was represented in Fig. 3. GC–MS analysis of C. acutum latex observed the chemical constituents such as lupeol, hexadecanoic acid, neophytadiene, octadecanoic acid and phytol were found with percentages of 15.36 %, 10.72 %, 9.15 %, 8.78 %, and 6.51 %, respectively (Soliman et al., 2022).
Group
Compound name
Molecular formula
Retention time (min)
Peak Area (%)
Medical applications
Alkene
1-Hexacosene
C26H52
34.306
0.145
Antibacterial (Rani et al., 2019)
Cholestanoid
17-(1,5- Dimethylhexyl)-10,13-dimethyl-2
C27H46O
38.780
0.18
Antioxidant activity (Nyalo et al., 2023)
Dioxolanes
1,3-Dioxolane, 4-methyl-2-pentadecyl
C21H42O2
3.104
0.382
Antimicrobial activity (Küçük et al., 2011)
Diosgenin
7-Dehydrodiosgenin
C27H40O3
37.654
0.155
Antioxidant, Anti-inflammatory, Anticancer (Semwal et al., 2022)
Fatty acid
Methyl Palmitoleate
C17H32O2
14.929
0.119
−
Palmitic acid
C16H32O2
25.998
0.988
Anticancer, Cardiovascular disease (Mancini et al., 2015)
Methyl (Z)- 5,11,14,17- eicosatetraenoate
C21H34O2
17.383
0.130
−
Octadecanoic acid
C18H36O2
18.714
0.751
Hypocholesterolemic activity (Selvaraju et al., 2021)
Myristic acid
C14H28O2
12.490
0.141
Larvicidal and Repellent activity (Chen et al., 2019)
Methyl linoleate
C19H34O2
17.266
0.147
Antioxidant activity (Davey et al., 2000)
L-Ascorbic acid
1-(+)-Ascorbic acid 2,6-dihexadecanoate
C38H68O8
15.336
8.129
Antioxidant activity (Babouongolo et al., 2021)
Phytosterol
17-(1,5-Dimethyl-3-phenylthiohex-4-enyl) −4,4,10,13,14-pentamethyl 2,3,4,5,6,7,10,11,12-
C36H54OS
47.067
0.593
Anticancer (Suttiarporn et al., 2015)
Campesterol
C28H48O
41.252
5.243
Anti-inflammatory and Cytotoxic activity (Bagewadi et al., 2019)
Phenol
Phenol, 2,6-dimethoxy-4-(2-propenyl)-
C11H14O3
11.909
0.108
Antioxidant activity (Molan et al., 2012)
Steroid
14-Dimethyl-5 alpha-ergosta-8
C30H50O
42.870
0.430
Anti-inflammatory, Anticancer (Rasheed & Qasim, 2013)
Pregn-5-en-20-one,12-(acetyloxy)-3,8,14
C23H34O6
38.302
0.136
−
Terpenoid
Cholesta-22,24-dien-5-ol,4,4-dimethyl-
C29H48O
41.877
3.482
−
Lupeol
C30H50O
46.326
13.061
Anti-inflammatory (Tsai et al., 2016)
Megastigmatrienone
C13H18O
11.065
0.116
Antioxidant activity (Kyslychenko et al., 2010)
Lupenone
C30H48O
45.271
0.306
Anti-inflammatory (Xu et al., 2020)
Beta-Amyrin
C30H50O
44.767
10.614
Antibacterial activity, Anti-inflammatory activity (Dash et al., 2023)
Phytol
C20H40O
17.572
1.455
Anti-inflammatory, Antimicrobial activity (Bagewadi et al., 2019)
Structure of the bioactive compounds obtained in the methanolic extract of Cynanchum tunicatum through GC–MS analysis.
The GC–MS analysis of Aristolochia tagala leaf extracts revealed the presence of 42 compounds across various solvents such as petroleum ether, chloroform, ethyl acetate, methanol, and hydro-alcoholic (Mariyammal et al., 2023). Olivia et al., utilized GC–MS analysis to identify twenty-three bioactive compounds in the hydromethanolic fraction of Hibiscus asper leaves such as 9,12,15-octadecatrien-1-ol, n-Hexadecanoic acid, octadecatrienol acid, methyl palmitate and phytol were significant phytoconstituents (Olivia et al., 2021). Lupeol and phytosterols possess various properties like antifungal, anti-inflammatory, antibacterial, anti-tumor, antioxidant and anti-ulcerative effects which plays their multifunctional biological roles (Ito et al., 2017). The GC–MS analyses showed preliminary insights into the bioactive components and evidenced the pharmaceutical applications for further research and drug discovery.
3.6 HPTLC profiling
In C. tunicatum the secondary metabolites were separated on HPTLC plates using various mobile phases such as Ethyl acetate: Methanol: Water (20:3:2), Ethyl acetate: Methanol: Formic acid: Water (20:3:1:2), Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2) and Toluene: Acetone: formic acid (4.5:4.5:1) for alkaloid, flavonoid, phenols and terpenoids respectively.
HPTLC Fingerprinting of alkaloid evidenced eight spots with their corresponding ascending order of Rf value of 0.032, 0.224, 0.302, 0.474, 0.597, 0.669,0.776, 0.976 in 2.5 µL of methanolic extract. Spot 1, 2, 3, and 5 were identified as strychnine (Anti-inflammatory, anticancer, hepatoprotective, antioxidant, cardio protective, antidepressant, antidote for snakebite), colchicine (decreases inflammatory cytokines) (Senguttuvan & Subramaniam, 2016). While in flavonoid showed eight bands in 2.5 µL of methanolic extract with corresponding Rf values were 0.048, 0.082, 0.310, 0.456, 0.684, 0.813, 0.890 and 0.966. Whereas in 5 µL of extracts, 6 bands were noted, and its corresponding Rf values were 0.076, 0.460, 0.703, 0.834, 0.900, 0.971.
Phenol showed three bands with respective Rf values were 0.031, 0.258, 0.813 in 2.5 µL of methanol extracts and eight bands formed with corresponding Rf values were 0.032, 0.090, 0.227, 0.448, 0.489, 0.785, 0.850, 0.961 in 5 µL of extract. In terpenoid, nine bands have appeared with the corresponding Rf values were 0.045, 0.113, 0.173, 0.192, 0.285, 0.398, 0.463, 0.553 and 0.629 in 2.5 µL of methanol extracts while in 5 µL of extract, ten bands with Rf values were 0.050, 0.118, 0.176, 0.285, 0.344, 0.402, 0.469, 0.561, 0.635 and 0.745 were obtained. The alkaloids, flavonoids, phenols and terpenoids were identified and quantified by comparing them with standards like colchicine, quercetin, gallic acid and oleanolic acid, respectively (Fig. 4). These bioactive components were reported strong antioxidant, antimicrobial, anti-inflammatory, anticancer, cardioprotective, anti-cardiovascular disease, antihemostatic, and chemopreventive activities (Deepika & Maurya, 2022). The metabolic screening showed that the primary constituents of the C. tunicatum extract comprised alkaloids, flavonoids, phenolic and terpenoids compounds.
TLC Profiles of Cynanchum tunicatum. a) Alkaloid at visible light b) Alkaloid at 366 nm c) Alkaloid at 254 nm d) Flavonoid at visible light e) Flavonoid at 366 nm f) Flavonoid at 254 nm g) Phenol at visible light h) Phenol at 366 nm i) Phenol at 254 nm j) Terpenoid at visible light k) Terpenoid at 366 nm l) Terpenoid at 254 nm.
4 Conclusion
In conclusion, the lacuna of requisite research in phytoconstituents of Cynanchum tunicatum influenced the spectrum of secondary metabolites, such as alkaloids, flavonoids, terpenoids and phenolic compounds were identified. In GCMS analysis methanolic extract of C. tunicatum showed 22 compounds were used for various therapeutic applications such as hypocholesterolemia, cancer, cardiovascular diseases, anti-ulcerative, antimicrobial, larvicidal and repellent activity. The phytochemicals identified using HPTLC fingerprinting were greatly beneficial for anti-inflammatory, anticancer, hepatoprotective, antioxidant, cardio protective, antidepressant, antidote for snakebite, and decreases inflammatory cytokines. Methanol extract of C. tunicatum evidenced potential in antioxidant and anti-inflammatory activities than the other extracts. Overall, the findings of this study of C. tunicatum as the major source of bioactive compounds with promising pharmacological activities suggested for further drug discovery from these identified compounds.
CRediT authorship contribution statement
Deepika Krishnamoorthy: Writing – original draft, Methodology, Investigation. Amutha Swaminathan: Writing – review & editing, Supervision, Methodology, Data curation, Conceptualization. Amal Mohamed AlGarawi: Writing – review & editing, Validation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Lavanya Nallasamy: Writing – review & editing, Validation, Methodology, Formal analysis, Data curation. Girija Sangari Murugavelu: Writing – review & editing, Formal analysis. Swarna Lakshmi Selvaraj: Writing – review & editing, Methodology, Formal analysis, Data curation.
Acknowledgment
I would like to thank the Department of Botany and CNR Rao Laboratory and Advanced Research Laboratory, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India. The authors extend their appreciation to the Researchers supporting project number (RSPD2024R931) King Saud University, Riyadh, Saudi Arabia.
Funding
This work was supported by the Researchers supporting project number (RSPD2024R931) King Saud University, Riyadh, Saudi Arabia.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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