Spectroscopic Analysis of Bioactive Compounds from Latex of Calotropis gigantea L. and an Evaluation of Its Biological Activities

Abstract

The current research investigation aimed to screen the bioactive compounds in the latex of Calotropis gigantea L. and evaluate its antibacterial and antioxidant properties towards clinical applications. The chemical moiety and volatile compounds of the latex of C. gigantea were detected by UV–Vis spectroscopy, FT-IR, and GC–MS analysis. The antibacterial activity was assessed using wound-inducing pathogens by well diffusion method. In addition, the antioxidant properties were determined through DPPH, ABTS, and FRAP methods. The functional groups of O–H stretch, diketonic bonds, C–O, C–N, O–C bonds, and consecutive C–H bonds were observed in the latex of C. gigantea. The major bioactive compounds were 5H-3,5a-Epoxynaphth[2,1-c]oxepin, Cholesta-5-en-3-ol, 24-propylidene-, dodecane, Lup-20(29)-Ene-3,28-Diol, (3.Beta)-, Veridiflorol, and Lanosta-8,24-dien-3-ol, acetatate, (3.beta.). Oxazole derivatives were found in the latex of C. gigantea, proved by GC–MS analysis. The aqueous-mixed latex exhibited maximum antioxidant activity as compared to methanol-mixed latex. Aqueous-mixed latex and methanol-mixed latex inhibited the growth of K. pneumoniae, P. mirabilis, S. pyogenes, Micrococcus spp., S. aureus, P. aeruginosa, and E. coli. The present study clearly reveals that latex of C. gigantea has rich bioactive compounds with significant biological activities, and can be employed to produce a novel herbal formulation against wound-inducing pathogens.

1. Introduction

Latex from plants has rich amount of phytochemicals, which are used to cure various diseases [1]. Latex is a milky fluid which contains proteins, alkaloids, and other phytometabolites. The colors of the latex differ from plant to plant, where the colors are white, yellow, orange, etc. [2]. Latex from plants shows antifungal, antihelminthic, antioxidant, anticancer, and antibacterial activities [2]. Ficus carica latex shows antimicrobial activity against human pathogens [3]. Latex proteins of Calotropis procera were used to treat the oral mucositis [4]. Latex of Jatropha neopauciflora Pax shows antimicrobial, wound-healing, and anti-inflammatory activities [5]. Sumathi et al. [6] investigated the antimicrobial properties of the latex of Euphorbia antiquorum. Prastiyanto et al. [7] stated the antibacterial potential of J. curcas, J. gossypilofia Linn., and J. multifida latex against Klebseilla pneumonia and E. coli. Using Havea brasiliensis, the rubber latex was extracted and was analyzed for its tissue and bone regeneration process [8].

C. gigantea is a flowering plant which belongs to the Apocynaceae family [9]. It is grown in many countries such as India, Cambodia, Vietnam, Thailand, Pakistan, and Nepal. The latex of C. gigantea alone can cure leprosy, cough, scabies, asthma, piles, etc. The phytochemicals, namely, terpenes, flavonoids, alkaloids, and pregnanes, are present in the latex of C. gigantea [10]. The present study was focused on screening and characterizing bioactive compounds from latex of C. gigantea and assessing their biological activities.

2. Materials and Methods

2.1. Collection of Latex, Preparation, and Phytochemical Screening

The fresh latex was carefully collected from the aerial parts of C. gigantea from Nakshatra garden, PSG College of Arts & Science, Coimbatore-14, India. Later, the latex was transferred to clean 1 mL Eppendorf tubes in an aseptic condition. As the latex coagulated property, it was slightly agitated after collection.

Briefly, 500 µL of different solvents such as petroleum ether, hexane, ethanol, methanol, and water were mixed well with 500 µL of C. gigantea latex. The mixture was centrifuged at 2000 rpm. The supernatants were stored in tubes at −4 °C. The phytochemicals were examined using the standard protocols [11,12].

2.2. UV–Visible Spectrophotometric Analysis

Different solvent mixtures of C. gigantea latex were analyzed using a UV–visible spectrophotometer (Shimadzu UV-1601 spectrophotometer) under the wavelength range from 200 to 800 nm. During analysis, the mixture was centrifuged at 2000 rpm for 15 min.

2.3. Analysis of Functional Groups

FTIR analysis was carried out using an FTIR spectrophotometer (FTIR (Model: Shimadzu; Miracle 10)), which identifies the functional groups present in the latex of C. gigantea. The latex was mixed with KBr powder and form as a disc. Then, it was analyzed and scanned from 4000 to 400 cm⁻¹.

2.4. Analysis of Phytoconstituents by Gas Chromatography/Mass Spectrometry

Gas chromatography–mass spectrometry (GC–MS) was conducted with the instrument CH-GCMSMS-02. The GC–MS instrument was supplied with a narrow bore (30 m length, 0.25 mm internal diameter, and 0.25 μm film thickness) for chromatographic separation (capillary standard nonpolar column). The atomizer temperature was optimized at 250 °C, with an atomizer volume of 1 μL in connected mode. Helium was the carrier gas, which flowed at 1 mL/min. The temperature of the GC oven program was optimized with a starting temperature of 70 °C, carried for 2 min, elevated at 10 °C/min to 250 °C, and then held for 5 min. In electron ionization (EI) mode, the mass spectrometer was operated with an ionization energy of 70 eV. The mass scan range was positioned between 50 and 800 m/z, and the electron beam source temperature was restored at 230 °C [13]. The components present in the extract were identified based on their retention times and mass spectra obtained from GC–MS analysis.

3. Determination of Biological Activities

3.1. Analysis of Antioxidant Properties

2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic)acid (ABTS) and ferric reducing antioxidant power (FRAP) assay were employed to determine the antioxidant activities of methanol- and aqueous-mixed latex of C. gigantea. Three replications were used for this investigation. The protocols for antioxidant assays were followed according to Shahinuzzaman et al. [14]. All results were measured in triplicate and expressed as means ± standard deviation (SD).

3.2. Analysis of Antibacterial Activity Using Well Diffusion Method

Klebsiella pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Micrococcus spp., Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were used for antibacterial activity. The cultures were subcultured and inoculated into the nutrient broth and kept in a shaker for 18 h at 37 °C. Bacterial cultures were swabbed using sterilized cotton swabs on Mueller–Hinton agar plates and wells were created by a sterilized well-cutter. Different concentrations of methanol- and aqueous-mixed latex of C. gigantea were prepared, and 60 µL, 80 µL, 100 µL, and 120 µL were poured to the corresponding wells. Antibiotic tetracycline was employed as the positive control. After that, the plates were incubated at 37 °C overnight. The zone of inhibition was measured after incubation. Three replications were maintained for this investigation. The mean values were calculated and expressed as mean ± SD.

4. Result and Discussion

4.1. Analysis of Phytochemicals

Table 1. Qualitative analysis of phytochemicals in the latex of C. gigantea.

S. No	Metabolites	Solvents
		Petroleum-Ether-Mixed Latex	Hexane-Mixed Latex	Ethanol Mixed-Latex	Methanol-Mixed Latex	Aqueous-Mixed Latex
1.	Alkaloids	−	−	+	+	−
2.	Flavonoids	−	−	−	−	−
3.	Sterols	−	−	−	−	−
4.	Terpenoids	−	−	+	+	−
5.	Anthraquinone	+	+	+	+	+
6.	Anthocyanin	−	−	−	−	−
7.	Proteins	−	−	−	−	−
8.	Phenolic compounds	−	−	−	−	−
9.	Quinones	−	−	−	−	−
10.	Carbohydrates	+	+	+	+	+
11.	Tannin	−	−	−	−	−
12.	Saponins	+	+	−	−	+
13.	Cardiac glycosides	−	−	+	+	−
14.	Glycoside’s test	−	−	−	−	−
15.	Lignin	−	−	−	−	−
16.	Coumarins	−	−	−	−	−
17.	Volatile oils	−	−	−	−	−

Note: “+” denotes “present”; “−” denotes “absent”.

determines the qualitative analysis of phytochemicals in the latex of C. gigantea. Alkaloids, terpenoids, anthraquinone, carbohydrates, and cardiac glycosides were present in both ethanol-mixed latex and methanol-mixed latex. The aqueous-mixed latex shows the anthraquinone, carbohydrates, and saponins. Petroleum-ether-mixed latex and hexane-mixed latex have anthraquinone, carbohydrates, and saponins. Saratha and Subramanian [15] studied the phytochemicals in C. gigantea latex extract and reported the presence of alkaloids, tannin, flavonoids, saponins, cardiac glycosides, terpenoids, amino acid, and steroids.

4.2. UV–Visible Spectrophotometric Analysis

Figure 1 displays the UV–visible spectra of C. gigantea latex used to identify the phytoconstituents occurring in the latex. In addition, it was performed to determine the phytomolecules containing chromophores and aromatic rings, σ-bonds, π-bonds, and lone pairs of electrons [16].

4.3. Analysis of Functional Groups

Different peaks, such as 3924, 1635, 1242, 1095, 686, 601, and 486 cm⁻¹, are observed for petroleum-ether-mixed latex of C. gigantea, which corresponds to functional groups of aliphatic aldehyde, C=C stretch, and C–H bending, respectively (Figure 2). Hexane-mixed latex of C. gigantea shows peaks at 3371, 1635, 1381, 1242, 1095, 605, and 470 cm⁻¹ that refer to O–H stretch, consecutive C=C stretch, and consecutive C–H bending/deformation, respectively (Figure 3). In the ethanol-mixed latex of C. gigantea, ten different peaks are observed, such as 3356, 2978, 2893, 1643, 1381, 1273, 1041, 879, 686, and 478 cm⁻¹, which indicate O–H stretch, consecutive C–H stretch, C=O stretch, and consecutive C–H bending, respectively (Figure 4).The methanol-mixed latex of C. gigantea displays different peaks at 1635, 1118, 1026, 725, 686, and 462 cm⁻¹, which indicate functional groups such as C=C stretch, C–H stretch, and consecutive C–H bending, respectively (Figure 5). Various peaks such as 3348, 1635, 1242, 686, 601, and 478 cm⁻¹, in aqueous mixed C. gigantea latex represent the functional groups of O–H stretch, diketonic bonds, C–O, C–N, O–C bonds, and consecutive C–H bonds, respectively (Figure 6). Similarly, Pradeepkumar et al. [17] carried out FTIR analysis for the latex of C. gigantea and reported the presence of functional groups such as O–H stretch, diketonic bonds, and C–N bonds. Sonia et al. [18] reported the occurrence of N–H stretch, C–H stretch, C–C stretch, C=C stretch, and C–H bending in C. gigantea flower latex (methanolic extract). FTIR analysis was evaluated for E. characias rubber latex, where peaks such as 1664 and 835 were observed, which correspond to C=C stretching and C–C bending, respectively [19]. FTIR analysis was conducted for Pergularia tomentosa latex, and different functional groups, namely, O–H stretch, N–H stretch, C=C stretch, C–H bending, and C–C–C deformation, were identified [20].

4.4. Analysis of Phytoconstituents by GC–MS

4.4.1. GC–MS Analysis of Aqueous-Mixed Latex of C. gigantea

More than 250 compounds were identified in aqueous-mixed latex of C. gigantea by gas chromatography/mass spectrometry (Figure 7 and

Table 2. GC–MS analysis of aqueous-mixed latex of C. gigantea.

S. No	Retention Time	Compound
1.	3.1164	Acetic acid
2.	3.2222	Diisopropylamine
3.	3.6468	n-Propyl acetate
4.	3.6943	Paraldehyde
5.	3.9246	Benzoic acid, 4-(1-methylpropyl)oxy-, methyl ester
6.	4.0701	4-Chloro-5-[[2 diethylaminoethyl]amino]pyridazin-3[2H]-one
7.	4.0773	2-Propenamide, N-[2-(diethylamino)ethyl]-N,2-dimethyl-
8.	4.1952	3(2H)-Furanone, dihydro-5-methyl-
9.	4.3936	Thiocyanic acid, ethyl ester
10.	4.4954	3-Methyl-6-ethyl--2,4-dioxadecane
11.	4.4957	2-Butenedioic acid (Z)-, dimethyl ester
12.	4.6320	Butanoic acid, 3-methyl-
13.	4.9677	2-Propenal
14.	5.0975	3,5,7,8-Tetrahydro-4,6-pteridinedione
15.	5.1014	Silane, (2-methoxyphenyl)trimethyl-
16.	5.2457	2-Heptenoic acid, 4-nitrophenyl ester
17.	5.2464	Bicyclo[2.2.1]heptan-2-ol, 2-(2-cyclopenten-1-yl)-
18.	5.3218	3-Hepten-2-one, 3-methyl-
19.	5.4819	Oxime-, methoxy-phenyl-_
20.	5.5294	Thiazole, tetrahydro-
21.	5.5387	2,2′-Bithiazolidine
22.	5.6710	1-Pentamethyldisilanyl-4-trimethylsiloxybenzene
23.	5.8823	Nitroguanidine
24.	5.8836	Thiomorpholine
25.	5.9934	Methyl vinyl ketone
26.	6.0348	Cyclobutanone, 2,3,3,4-tetramethyl-
27.	6.1198	3-Pentenoic acid, 4-methyl-, methyl ester
28.	6.4138	Pyridine, 2-(1-methylethyl)-
29.	6.7693	2-Thiophenethiol
30.	6.9591	2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
31.	7.0853	2-Hydroxypentanediamide
32.	7.1416	Bicyclo[3.1.0]hexan-3-ol, 4-methyl-1-(1-methylethyl)-
33.	7.1760	alpha.-Phellandrene
34.	7.2121	2-Furancarboxylic acid, 2-methoxyethyl ester
35.	7.2822	Cyclotetrasiloxane, octamethyl-
36.	7.3176	6-Hepten-1-ol, 2-methyl-
37.	7.5330	1,4-Butanediamine, 2,3-dimethoxy-N,N,N′,N′-tetramethyl-, [S-R*,R*)]-
38.	7.6244	Propanoic acid, ethenyl ester
39.	7.6246	2-Pentenoic acid, 4-methyl-
40.	7.6250	2-Butenoic acid, ethyl ester, (E)-
41.	7.6500	Pentane, 2,2,4,4-tetramethyl-3-methoxy-
42.	7.7127	1,3-Cyclohexadiene, 1-methyl-4-(1-methylethyl)-
43.	7.7757	Hydrazine, 1,1-dibutyl-
44.	7.8171	1,3-Cyclohexadiene, 1-methyl-4-(1-methylethyl)-
45.	7.9027	Benzene, 1-methyl-3-(1-methylethyl)-
46.	7.9923	2-Hydroxyquinine
47.	7.9936	D-Limonene
48.	8.0039	Hydroxylamine, O-(phenylmethyl)-
49.	8.0363	m-Aminophenylacetylene
50.	8.0411	Benzene, 1,2,4,5-tetramethyl-
51.	8.1266	D-Limonene
52.	8.1698	3-Acetoxy-3-hydroxypropionic acid, methyl ester
53.	8.3868	Bicyclo[3.1.0]hex-2-ene, 2-methyl-5-(1-methylethyl)-
54.	8.4197	Furan, 2-methyl-5-(methylthio)-
55.	8.4611	Bicyclo[3.1.0]hex-2-ene, 2-methyl-5-(1-methylethyl)-
56.	8.4768	2H-1,2,4-Oxadiazine-3,6(4H,5H)-dione, 4,5-dimethyl-2-phenyl-
57.	8.5869	1-Amino-2,6-dimethylpiperidine
58.	8.6649	3-Carene
59.	8.7552	Thymine
60.	8.8170	Cyclotrisiloxane, hexamethyl-
61.	8.8774	Benzofuran-5,6-diol-3-one, 2-benzylidene-
62.	8.9324	.gamma.-Terpinene
63.	8.9842	2-Isopropenylthiazole
64.	9.3303	Cyclotrisiloxane, hexamethyl-
65.	9.3745	2-Furanmethanol, 5-ethenyltetrahydro-.alpha.,.alpha.,5-trimethyl-, cis-
66.	9.3746	trans-Linalool oxide (furanoid)
67.	9.4544	1,3-Cyclohexadiene, 1-methyl-4-(1-methylethyl)-
68.	9.4594	1,3-Cyclohexadiene, 1-methyl-4-(1-methylethyl)-
69.	9.5043	Benzene, 1-ethenyl-3,5-dimethyl-
70.	9.5073	Benzaldehyde, 4-(1-phenyl-2-propenyloxy)-
71.	9.7270	(+)-4-Carene
72.	9.7498	Cyclotrisiloxane, hexamethyl-
73.	9.7837	Benzene, 4-ethenyl-1,2-dimethyl-
74.	9.9367	3-Methyl-5-nonylpyrrolizidine
75.	9.9521	Dideoxy-dimethylene-iditol
76.	10.0442	1,3-Benzenediol, monobenzoate
77.	10.3802	1H-Imidazo[4,5-b]pyridine
78.	10.5562	4-Ethylbenzamide
79.	10.6038	Maltol
80.	10.7661	Methyl 2-O-methyl-.beta.-D-xylopyranoside
81.	10.8566	Benzoic acid, hydrazide
82.	10.8595	1,3-Cyclohexadiene, 1,3,5,5-tetramethyl-
83.	10.8951	Benzene, 1-ethyl-3,5-dimethyl-
84.	10.9589	trans-(2-Chlorovinyl)dimethylethoxysilane
85.	11.1955	1,3-Cyclopentadiene, 1,2,3,4,5-pentamethyl-
86.	11.1976	N-Formyl-1-phenylethylamine, N- (pentafluoropropionyl)-
87.	11.2112	1,3-Cyclohexadiene, 1,3,5,5-tetramethyl-
88.	11.2344	2,4,6-Octatriene, 2,6-dimethyl-, (E,Z)-
89.	11.4400	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
90.	11.5297	L-Alanyl-L-norleucine, N-dimethylaminomethylene-,methylester
91.	11.7208	1-Methyl-5-fluorouracil
92.	11.7286	Ethyl hydrogen succinate
93.	12.0331	2,4-Pentanedione, 1,1,1,5,5,5-hexafluoro-
94.	12.1718	Bis(hexahydro-7a-methyl-3-oxo-3H-pyrrolizin-5-yl)ether
95.	12.2723	3,4-Hexanedione, 1 (phenylmethoxy)-
96.	12.2892	1-(Hexahydropyrrolizin-3-ylidene)-3,3-dimethyl-butan-2-one
97.	12.3125	Thiazolidine, 2-isobutyl-
98.	12.5429	1,5-Heptadiene, 3,3,6-trimethyl-
99.	12.6079	L-.alpha.-Terpineol
100.	12.8687	1,3,2-Oxazaborolidine-4-carboxylic acid, 2-butyl-5-methyl-, methyl ester, cis-
101.	13.1899	1,4:3,6-Dianhydro-.alpha.-d-glucopyranose
102.	13.2456	Ethanol, 2-phenoxy-
103.	13.3101	Dimethyl sulfone
104.	13.3444	6-Chlorohexanoic acid, 4-isopropylphenyl ester
105.	13.3724	1,3-Benzenediacetonitrile
106.	13.4215	Cyclohexanecarboxylic acid, 4-propyl-, 4-ethoxyphenyl ester, trans-
107.	13.4450	Bicyclo[2.2.1]heptane, 2-chloro-1,7,7-trimethyl-,exo-
108.	13.4452	Bicyclo[2.2.1]heptane, 2-chloro-1,7,7-trimethyl-,exo-
109.	13.5029	2-Methoxy-3-methyl-butyric acid, methyl ester
110.	13.5817	2-Chloro-4,6-difluoro-pyrimidine
111.	13.6006	Dinocap
112.	13.8528	1-Bromopyrene
113.	14.1003	2,6-Octadiene, 4,5-dimethyl-
114.	14.1213	Geranyl bromide
115.	14.6500	l-Norvaline, N-allyloxycarbonyl-, isobutyl ester
116.	14.8145	Benzoic acid, 3-methyl-, methyl ester
117.	14.8153	Benzeneacetic acid
118.	14.8806	(-)-O-Acetylmalic anhydride
119.	15.0060	l-Leucine. N-(3-fluorobenzoyl)-, hexyl ester
120.	15.4630	1,3-Benzodioxole, 5-propyl-
121.	15.4732	3,4-(Methylenedioxy)toluene
122.	15.5623	Cyclobutane, 1,3-diisopropenyl-, trans
123.	15.7477	Thymol
124.	16.0172	2-Methoxy-4-vinylphenol
125.	16.2591	1-Naphthoic acid, 4-chloro-2-methylphenyl ester
126.	17.3087	Succinic acid, di(2-fluoroethyl) ester
127.	18.1799	Cyclopentasiloxane, decamethyl-
128.	18.4063	Cyclopropanecarboxylic acid, pent-2-en-4-ynyl ester
129.	18.4212	1,4-Benzenediol, 2-methoxy-
130.	18.6433	Isoquinoline, 1,2,3,4-tetrahydro-6,7-dihydroxy-1-methyl-
131.	18.9219	3-Acetoxy-2-methyl-pyran-4-one
132.	18.9566	Benzenemethanol, .alpha.-methyl-.alpha.-(1-methyl-2-propenyl)-
133.	19.5578	2-tert-Butyl-3,4,5,6-tetrahydropyridine
134.	19.9121	Ethanone, 1-(3-hydroxy-4-methoxyphenyl)-
135.	20.2332	beta.-D-Glucopyranose, 1,6-anhydro-
136.	20.2439	Pentanoic acid, 5-hydroxy-, 2,4-di-t-butylphenyl esters
137.	20.4522	Benzoic acid, 4-ethoxy-, ethyl ester
138.	20.4815	2,5-Dihydroxy-4-methoxyacetophenone
139.	20.6307	Propan-2-one, 1-(4-isopropoxy-3-methoxyphenyl)-
140.	20.7563	4′-(Trifluoromethyl)acetophenone
141.	20.7858	Nonanedioic acid, dimethyl ester
142.	20.9204	Pyrido[2,3-b]indole, 6-amino-
143.	20.9761	Glutaric acid, 2-pentyl propyl ester
144.	21.0824	Phenol, 4-ethenyl-2,6-dimethoxy-
145.	21.3600	3-Hydroxy-4-methoxybenzoic acid
146.	21.5168	Butamben
147.	22.1123	3-Phenyl-4-hydroxyacetophenone
148.	22.1584	4-Pyridinol, 2,6-dimethyl-3-[methylthio]-
149.	22.3544	2,5-Piperazinedione, 3-methyl-6-(1-methylethyl)-
150.	23.0161	4′-(1-Pyrroyl)acetophenone
151.	23.1652	L-Proline, N-(hexanoyl)-, pentyl ester
152.	23.2674	2H-1-Benzopyran-3-carbonitrile, 4-methyl-2-oxo-
153.	23.3523	9H-Xanthene
154.	23.5004	(3S,6S)-3-Butyl-6-methylpiperazine-2,5-dione
155.	23.6610	3,6-Diisopropylpiperazin-2,5-dione
156.	24.3079	Cyclo(L-prolyl-L-valine)
157.	24.5477	2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl)-,methyl ester
158.	24.6445	Valeric acid, 2-biphenyl ester
159.	24.7733	Phthalic acid, cyclohexyl isohexyl ester
160.	24.7733	Phthalic acid, propyl tridec-2-yn-1-yl ester
161.	24.8451	Cyclotetradecane
162.	24.9312	Tetradecanoic acid, 10,13-dimethyl-, methyl ester
163.	25.5461	Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-
164.	25.7591	4-Methylumbelliferyl laurate
165.	25.7692	Phenol, 4-[(5,6,7,8-tetrahydro-1,3-dioxolo[4,5-g]isoquinolin-5-yl)methyl]-, (R)-
166.	25.8003	n-Hexadecanoic acid
167.	25.8927	Propane-1,1-diol dipropanoate
168.	25.8976	Heptadecanoic acid, methyl ester
169.	26.1829	Hexadecanoic acid, 14-methyl-, methyl ester
170.	26.2367	Methyl 8-heptadecenoate
171.	26.3061	Methyl 8-heptadecenoate
172.	26.6748	Dodecanamide, N-(2-hydroxyethyl)-
173.	26.9179	1,13-Tetradecadiene
174.	26.9181	5-Cyclohexyl-1-pentene
175.	27.1865	1-Hexadecanol
176.	27.5357	Phytol
177.	27.6157	2-Furancarboxamide, N-ethyl-
178.	27.8322	Dodecanamide, N-(2-hydroxyethyl)-
179.	27.8570	cis-13-Octadecenoic acid
180.	27.8887	2-Cyano-5-(3-fluorophenyl)pyrimidine
181.	27.9256	Benzene, (1-methylbutyl)-
182.	28.0760	2-Propanamine, N,N′-methanetetraylbis-
183.	28.2097	2-Phenylacetic acid, 2,2,2-trifluoroethyl ester
184.	28.6594	(Z)-2-(pentadec-8-en-1-yl)-4,5-dihydrooxazole
185.	28.6741	(Z)-2-(pentadec-8-en-1-yl)-4,5-dihydrooxazole
186.	28.7725	Cyclohexanethiol, 2,5-dimethyl-, acetate
187.	28.8193	Pivalic acid, 2-methylpropyl ester
188.	29.0173	Cyclohexanethiol, 2,5-dimethyl-, acetate
189.	29.0827	Cyclohexanepropanol-
190.	29.0831	5-Methyl-4-hexene-1-yl acetate
191.	29.1350	Cyclohexanethiol, 2,5-dimethyl-, acetate
192.	29.2850	Cyclohexanethiol, 2,5-dimethyl-, acetate
193.	29.3103	3-Chloropropionic acid, heptadecyl ester
194.	29.3207	Cyclohexane, tetracosyl-
195.	29.6797	Oxazole, 2-(8Z)-8-heptadecen-1-yl-4,5-dihydro-
196.	29.7709	Octyl tiglate
197.	29.9002	1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-,(E,E)-
198.	30.1936	Benzene, 5-(2-isothiocyanatoethyl)-1,2,3-trimethoxy-
199.	30.2872	1-Heneicosanol
200.	30.3570	Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-
201.	30.4001	1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-,(E,E)-
202.	30.6341	(E)-1-(6,10-Dimethylundec-5-en-2-yl)-4-methylbenzene
203.	30.6343	(E)-1-(6,10-Dimethylundec-5-en-2-yl)-4-methylbenzene
204.	30.7528	p-Camphorene
205.	30.7534	(E)-1-(6,10-Dimethylundec-5-en-2-yl)-4-methylbenzene
206.	30.7593	1-Ethyl-1,3,3-trimethyl-5-(1-methyl-3-propanal)indan
207.	30.8610	Squalene
208.	30.8621	Supraene
209.	30.8721	Hexane, 3,4-bis(1,1-dimethylethyl)-2,2,5,5-tetramethyl-
210.	30.9253	1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-,(E,E)-
211.	31.0366	(E,E,E)-3,7,11,15-Tetramethylhexadeca- 1,3,6,10,14-pentaene
212.	31.0368	(E)-1-(6,10-Dimethylundec-5-en-2-yl)-4-methylbenzene
213.	31.1347	Palmitoyl chloride
214.	31.2706	Glycidyl palmitate
215.	31.4125	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester
216.	31.5403	Isobutyl 2-(4-methylcyclohex-3-enyl)propan-2-ylcarbonate
217.	31.5760	Fenoterol, N-trifluoroacetyl-O,O,O,O-tetrakis(trimethylsilyl)deriv.
218.	31.5859	Tetracosamethyl-cyclododecasiloxane
219.	32.0501	trans-Geranylgeraniol
220.	32.1611	trans-Geranylgeraniol
221.	32.2627	Benzoic acid, hexadecyl ester
222.	32.2976	Supraene
223.	32.4260	(2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-tetraen-1-yl formate
224.	32.6000	L-Valine, N-(2-fluoro-5-trifluoromethylbenzoyl)-,isohexyl ester
225.	32.6258	Oleoyl chloride
226.	32.6261	9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester
227.	32.6605	6H-Dibenzo[b,d]pyran-6-one, 7,9-dihydroxy-3-methoxy-1-methyl-
228.	32.6607	9,10-Dihydro-10-phenyl-9,10-ethano-9-phosphaanthracene-11,12-dicarboxylic anhydride
229.	32.7174	1,2,3,4-Tetrahydro-3-(phenylacetamido)quinoline
230.	32.7381	Squalene
231.	32.7873	Isonipecotic acid, N-(3-phenylpropionyl)-, undecyl ester
232.	32.8123	Stearic acid chloride
233.	32.8338	Petasitene
234.	32.9963	Isophthalic acid, di(2-fluorophenyl) ester
235.	33.0156	Acetamide, 2-(1-benzimidazolyl)-
236.	33.0293	Octadecanoic acid, 2,3-dihydroxypropyl ester
237.	33.0835	Methyl-methoxy-hydroxymethyl-amine
238.	33.0864	5H-Tetrazol-5-amine
239.	33.1417	DL-Lactamide, N,N-dimethyl-, methyl ether
240.	33.4009	(2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-tetraen-1-yl formate
241.	33.7179	Pentyl palmitoleate
242.	33.7348	9-Hexadecenoic acid, octadecyl ester, (Z)-
243.	33.8473	(E,E)-7,11,15-Trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene
244.	34.2625	Tetradecanoic acid, octadecyl ester
245.	34.6215	Pyrido[2,3-d]pyrimidin-5(8H)-one, 2-methyl-4-phenyl-
246.	34.6353	9-Hexadecenoic acid, octadecyl ester, (Z)-
247.	34.8413	Benzimidazole, 1-(4-chloro-1-methyl-3-pyrazolyl)carbonyl-
248.	34.9489	Squalene
249.	35.0604	Squalene
250.	35.6490	2-Fluorobenzylamine, N,N-diheptyl
251.	35.6490	9-Hexadecenoic acid, octadecyl ester, (Z)-
252.	35.6538	9-Hexadecenoic acid, octadecyl ester, (Z)-
253.	35.8164	trans-Geranylgeraniol
254.	36.0085	1,6,10,14,18,22-Tetracosahexaen-3-ol, 2,6,10,15,19,23-hexamethyl-, (all-E)-(.+/−.)-
255.	36.0566	Silane, diethylheptyloxyoctadecyloxy-
256.	36.2410	Oxirane, 2-dimethyl-3-(3,7,12,16,20-pentamethyl-3,7,11,15,19-heneicosapentaenyl)-, (all-E)-
257.	36.4041	Oxirane, 2-dimethyl-3-(3,7,12,16,20-pentamethyl-3,7,11,15,19-heneicosapentaenyl)-, (all-E)-
258.	36.4347	Benzalphthalide
259.	36.8772	(2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-tetraen-1-yl formate
260.	36.8830	2,6,10,14-Hexadecatetraen-1-ol, 3,7,11,15-tetramethyl-, acetate, (E,E,E)-
261.	37.2283	Urs-12-en-3-ol, acetate, (3.beta.)-
262.	37.4453	Vitamin E
263.	37.4462	9,9′-Bi-9H-fluorene
264.	37.4947	Ethane, 1,2-diiodo-

). The following compoundswere prominently present in aqueous-mixed latex of C. gigantea: 1,3-Cyclohexadiene, 1-methyl-4-(1-methylethyl)- with retention time 7.71; D-Limonene with retention time 8.12; Bicyclo[3.1.0]hex-2-ene, 2-methyl-5-(1-methylethyl)- with retention time 8.38; Cyclotrisiloxane, hexamethyl- with retention time 8.81; 1,3-Cyclohexadiene, 1-methyl-4-(1-methylethyl)- with retention time 9.45;Bicyclo[2.2.1]heptane, 2-chloro-1,7,7-trimethyl, exo- with retention time 13.44; Methyl 8-heptadecenoate with retention time 26.23; Dodecanamide, N-(2-hydroxyethyl)- with retention time 26.67; (Z)-2-(pentadec-8-en-1-yl)-4,5-dihydrooxazole with retention time 28.65; Cyclohexanethiol, 2,5-dimethyl-, acetate with retention time 28.77; 1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl, (E,E)- with retention time 30.40; (E)-1-(6,10-Dimethylundec-5-en-2-yl)-4-methylbenzene with retention time 30.63; Squalene with retention time 30.86; 1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl-, (E,E)- with retention time 30.92; Supraene with retention time 32.29;trans-Geranylgeraniol with retention time 32.05; (2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-tetraen-1-yl formate with retention time 32.42; 9-Hexadecenoic acid, octadecyl ester, (Z)- with retention time 33.73; and Oxirane, 2,2-dimethyl-3-(3,7,12,16,20-pentamethyl-3,7,11,15,19-heneicosapentaenyl)-, (all-E)- with retention time 36.24. Cyclohexanethiol, 2,5-dimethyl-acetate, a sulfur-containing compound, has shown promising anticancer potential by inhibiting cancer cell growth and metastasis.

4.4.2. GC–MS Analysis of Methanol-Mixed Latex of C. gigantea

Thirty compounds were identified in aqueous-mixed latex of C. gigantea by gas chromatography/mass spectrometry (Figure 8 and

Table 3. GC–MS analysis of methanol-mixed latex of C. gigantea.

S. No	Retention Time	Compound
1.	3.546	2-methylbutyl acetate
2.	3.592	1-Butanol, 2-Methyl-,Acetate
3.	6.572	CIS-Sesquilavandulol
4.	7.483	Propene-3,3,3-D3
5.	7.693	4-Heptyn-2-ol
6.	18.620	Furo[3,4-D]-1,3-Dioxole-3A(4H)-Car
7.	20.180	Decane, 1-Chloro
8.	25.043	(4.Alpha.,10.Beta.,15s)-4-Carbomeo
9.	30.232	Tetradecanoic Acid, 12-Methyl
10.	30.413	Phthalic acid, 4-bromophenyl heptyl ester
11.	31.076	3,4-Hexanediol, 2,5-Dimethyl
12.	34.028	Tetradecanoic Acid, 12-Methyl-,
13.	41.510	1,5-Heptadiene, 2,6-Dimethyl-
14.	41.691	2z,6e-Farnesol
15.	46.408	1,9-Nonanediol, Dimethanesulfonate
16.	46.582	N-(4-Methylcyclohexyl)Acetamide, Cis-
17.	47.580	6,11-Dimethyl-2,6,10-Dodecatrien
18.	53.652	Pseduosarsasapogenin-5,20-Dien
19.	54.012	Cholest-5-En-3-Ol, 24-Propylidene-, (3.Beta
20.	54.248	1,6,10-Dodecatrien-3-Ol, 3,7,11-Tri
21.	54.358	Urs-12-En-28-Ol
22.	54.400	Cholesta-8,24-Dien-3-Ol, 4-Methyl-, (3.Beta
23.	55.418	1,6,10-Dodecatrien-3-Ol, 3,7,11-Trim
24.	55.678	Dehydrolinalool
25.	56.939	3-Decyn-2-Ol
26.	57.348	5H-3,5a-Epoxynaphth[2,1-C]Oxepin, Dodeca
27.	58.010	1-Heptatriacotanol
28.	58.313	Lup-20(29)-Ene-3,28-Diol,(3.Beta.)-
29.	58.624	Veridiflorol
30.	58.885	Lanosta-8,24-dien-3-ol, acetate,(3.beta.)-

). Cholesta-5-en-3-ol, 24-propylidene- with retention time 54.01, 5H-3,5a-Epoxynaphth[2,1-c]oxepin with retention time 57.34, Lup-20(29)-Ene-3,28-Diol, (3.Beta)- with retention time 58.31, Veridiflorol with retention time 58.62, and Lanosta-8,24-dien-3-ol, acetatate, (3.beta.)- with retention time 58.88 outstandingly occurred in methanol-mixed latex of C. gigantea. All the above phytochemicals show antibacterial, anti-inflammatory, antiviral, antifungal, and antioxidant properties. Sharma et al. [21] found 22 compounds in methanolic extract of C. gigantea (L.) WT Aiton latex through GC–MS analysis.

4.5. Antioxidant Activity

Table 4. Antioxidant analysis by DPPH method.

Concentrations (µL)	DPPH Assay (% of Inhibition)
	MC	AC
10	40.98 ± 1.47	65.57 ± 1.12
50	52.46 ± 1.69	68.03 ± 1.73
150	59.02 ± 1.92	81.15 ± 1.15
250	60.84 ± 1.78	82.75 ± 1.36
350	64.75 ± 1.04	83.61 ± 1.79
500	65.57 ± 1.84	85.25 ± 1.92
750	68.85 ± 1.23	86.35 ± 1.67
IC50	32.15 µg/mL	30.16 µg/mL

Note: MC: methanol-mixed latex of C. gigantea; AC: aqueous-mixed latex of C. gigantea.

,

Table 5. Antioxidant analysis by ABTS method.

Concentrations (µL)	DPPH Assay (% of Inhibition)
	MC	AC
10	42.23 ± 1.01	50.46 ± 2.89
50	50.35 ± 1.56	65.92 ± 3.58
150	57.90 ± 1.62	70.24 ± 2.13
250	59.73 ± 1.81	72.86 ± 1.45
350	60.64 ± 1.25	75.72 ± 2.54
500	63.46 ± 1.04	80.37 ± 2.12
750	65.74 ± 1.66	83.46 ± 1.45
IC50	40.14 µg/mL	38.22 µg/mL

Note: MC: methanol-mixed latex of C. gigantea; AC: aqueous-mixed latex of C. gigantea.

and

Table 6. Antioxidant analysis by FRAP method.

Concentrations (µL)	DPPH Assay (% of Inhibition)
	MC	AC
10	31.12 ± 1.89	49.35 ± 1.48
50	39.24 ± 1.71	54.81 ± 1.40
150	46.02 ± 1.33	59.13 ± 1.29
250	48.62 ± 1.67	61.75 ± 1.32
350	49.53 ± 1.29	64.61 ± 1.74
500	50.35 ± 1.57	69.59 ± 1.87
750	54.63 ± 1.05	73.14 ± 1.96
IC50	33.59 µg/mL	31.28 µg/mL

Note: MC: methanol-mixed latex of C. gigantea; AC: aqueous-mixed latex of C. gigantea.

summarizes the results of antioxidant properties of methanol-mixed latex and aqueous-mixed latex via DPPH, ABTS, and FRAP assay. The aqueous-mixed latex displays maximum antioxidant activity when compared to methanol-mixed latex, confirmed by all the methods. Shahinuzzaman et al. [14] investigated the in vitro antioxidant potential of Ficus carica L. latex. Bioactive metabolites in the latex of Croton lechleri were identified, andits antioxidant activity was investigated by De Marino et al. [22]. The antioxidant properties of Pergularia tomentosa latex extract were investigated using DPPH assay.

4.6. Antibacterial Activity

Aqueous-mixed latex and methanol-mixed latexinhibit the growth of K. pneumoniae, P. mirabilis, S. pyogenes, S. aureus, Micrococcus spp., P. aeruginosa, and E. coli. Figure 9 and Figure 10 show the antibacterial activity of aqueous-mixed latex and methanol-mixed latex. Similarly, Subramanian and Saratha [23] reported the antibacterial action of C. gigantea latex extract on selected pathogenic bacteria and concluded that the C. gigantea latex has good antibacterial properties. An investigation was performed by Kumar et al. [24] to determine the antimicrobial potential of C. gigantea latex against pathogenic microorganisms. They stated that C. gigantea latex has rich antimicrobial properties.

5. Conclusions

In conclusion, C. gigantea latex has various bioactive compounds, proved by phytochemical screening, FTIR, and GC–MS analysis. Phytochemical screening revealed the presence of alkaloids, terpenoids, anthraquinone, carbohydrates, and saponins in the latex. The following major bioactive compounds were present in the latex of C. gigantea: Cholesta-5-en-3-ol, 24-propylidene-, 5H-3,5a-Epoxynaphth[2,1-c]oxepin, dodecane, Lup-20(29)-Ene-3,28-Diol, (3.Beta)-, Veridiflorol and Lanosta-8,24-dien-3-ol, acetatate, (3.beta.), Oxazole derivatives, fatty acid derivatives, and sulfur-containing compounds. The latex of C. gigantea shows antioxidant and antibacterial activity. C. gigantea holds promise as a valuable bioactive resource for developing novel therapeutics and discovering new treatment modalities.