Comprehensive Phytochemical Analysis of Caesalpinea mimosoides: Comparative Evaluation of Leaf and Stem Extracts Using Multiple Solvents

INTRODUCTION

Plants are a rich source of therapeutic compounds with numerous applications in the pharmaceutical industry. Plant products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for brand-spanking-new drug leads due to the unequalled availability of chemical diversity. Medicinal plants are rich in bioactive components that are utilized to treat various human ailments. In recent years, there has been a growing awareness of the importance of medicinal plants. According to the World Health Organization (WHO), traditional plant-based medicines constitute the primary source of healthcare for more than 80% of the world’s population in developing and underprivileged nations.^[1,2] The WHO has made an effort to identify all internationally used medicinal plants and recognized over 20,000 species.^[3] Most people in society utilize these plants as herbal remedies or as pharmaceutical ingredients in contemporary medicine.^[4] Drugs derived from plants are readily available, less expensive, safe, and efficient, and have fewer side effects.^[5] The therapeutic importance of plants depends upon the type of phytochemical compounds they possess, showing various physiological effects on the human body. Hence, the phytochemical screening method can be employed to detect these compounds present in plants, which may be used as the basis for modern drug development.^[6] Caesalpinia mimosoides Lamk. (family Fabaceae; subfamily Caesalpiniaceae) is a small spiny tropical tree or climbing shrub distributed in several countries, such as China, India, Myanmar, and Thailand. It is known locally as “theemullu” or “kenchige”. Within the realm of traditional Indian healing practices, Caesalpinia mimosoides has been used for a long time to address various health concerns. The plant C. mimosoides has several traditional uses, including food and medicine.^[7] Plant phytochemicals have been reported to prevent several diseases, including cancer, cardiovascular disease, infection, and inflammation. The plant is known to possess antimicrobial,^[8,9] antioxidant,^[10] antiviral,^[11] anti-inflammatory, anticholinesterase, and neuroprotective properties, according to the literature,^[12] and has been shown to inhibit the viability of cervical carcinoma cell lines.^[13] Despite its longstanding use in Ayurveda and tribal medicine, C. mimosoides remains largely understudied. Although numerous studies have been conducted on these plant extracts to test various biological activities, and extracts identified as having specific properties have been analysed for their phytochemical constituents, most studies have focused on the phytochemical screening of one or two solvents, with some studies limited to a single solvent. In this regard, the present study will focus on comparative analysis of phytochemical evaluation of the most commonly used parts of the plants by traditional healers, like stem and leaves, with different solvent extraction, which can give scientific evidence for the use of the plant in various conditions, along with identification of the potential compounds for pharmaceutical evaluation as potential drug candidates.

MATERIAL AND METHODS

RESULTS

Phytochemical analysis using UPLC-MS/MS

A. Hexane extract –Leaf and stem

The Hexane extract of the leaves yielded ∼867 compounds, with >35 distinct peaks. Important phytochemical classes detected include triterpenoid saponins, sterols, polyphenols, and bicyclic hexapeptides.

Major active phytochemicals detected were Ruvoside, Celosin F, Ergosterol peroxidase, 5-Undecyl-1,3-benzodioxole, and Bicyclic peptide RAxix [Table 1, Figure 1].

Table 1: The major compounds identified from the hexane extract of leaf and stem of Caesalpinea mimosoides according to UHPLC-MS/MS.

Leaf extract	tR (min)	Compound name	Classification	Formula	Calculated mass	m/z	Extract mass	Mass accuracy (mDa)	% Base peak
	19.87	Ruvoside	Polyphenol	C30H46O9	550.31	568.34	550.31	0.6	5.30
	11.8	RA-Xix	Bicyclic hexapeptides	C44H57N6O9	812.41	445.16	812.40	-3.5	4.07
	25.94	Celosin F	Saphonins	C35H50O12	662.33	685.31	662.33	0.2	3.48
	22.16	Ergosterol peroxide	Sterols	C28H44O3	428.32	429.33	276.21	0.9	2.76
	13.26	5-Undecyl-1,3-benzodioxole	Polyphenols	C18H28O2	276.20	277.21	645.31	1.2	2.76
Stem extract	25.86	Celosin F	Saphonins	C35H50O12	662.33	663.33	662.33	1.4	5.2078
	19.05	Octadecanal	Bicyclic hexapeptides	C18H36O	268.27	173.10	268.27	1.3	5.439
	26.32	Purpurin	Sterols	C14H8O5	256.03	140.01	256.037	-0.2	3.3885
	6.93	Ginkgolid J	Sterols	C20H24O10	424.13	425.14	424.13	0.3	2.7392
	14.62	Atractyloside D	Polyphenols	C27H46O12	562.29	301.13	562.30	2.9	3.2692

tR: Retention time; m/z: Mass to charge ratio; UHPLC-MS: Ultra-high performance liquid chromatography-mass spectrometry; MS: Mass spectrometry.

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Figure 1: UHPLC-MS chromatogram of the hexane extract of the leaf of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: Ultra-high performance liquid chromatography-mass spectrometry.

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The hexane extract of the stem identified ∼438 compounds with 21 distinct peaks. Important phytochemical classes detected were similar to those found in leaf extracts, including triterpenoid saponins, sterols, polyphenols, and bicyclic hexapeptides.

However, active phytochemical compounds were celosin F, octadecanal, ginkgolid J, atractyloside D, and purpurin [Table 1, Figure 2].

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Figure 2: UHPLC-MS chromatogram of the hexane extract of the stem of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: Ultra-high performance liquid chromatography-mass spectrometry.

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B. Ethyl acetate extract: Leaf and stem

The ethyl acetate extract of the plant leaves identified ∼300 compounds, corresponding to 61 peaks. The majority of the phytochemicals belong to the class of alkaloids, glycosides, non-flavonoid polyphenols, triterpenoids, and steroids.

Among these, 3-phenylpyridine is the primary compound detected in around seven peaks of the LC-MS results. Other vital phytochemicals detected are cyanoside G, caffeic acid, 2-benzylidene succinic acid, and abruside A [Table 2, Figure 3].

Table 2: The major compounds identified from the ethyl acetate extract of leaf and stem of Caesalpinea mimosoides according to UHPLC–MS/MS.

Leaf extract	tR (min)	Compound name	Classification	Formula	Calculated mass	m/z	Extract mass	Mass accuracy (mDa)	% Base peak
	26.23	3- Phenylpyridine	Alkaloid	C11H9N	155.071	173.01	155.07	0.1	14.58
	16.02	Cynanoside G	Glycoside	C41H62O16	810.40	425.18	810.40	3.1	8.507
	26.52	Caffeic acid	Polyphenols	C9H8O4	180.04	128.98	180.04	1.1	7.164
	25.46	Abrusoside A	Glycoside	C36H54O10	646.37	685.33	646.37	0	5.129
	26.45	2-Benzylidene succinic acid	Polyphenols	C11H10O4	206.057	14199	206.05	-0.6	3.926
Stem extract	26.11	Phaginin I	Glycoside	C21H28O4	344.19	173.10	344.19	-0.5	5.995
	18.08	Cartormin	Terpenoids	C27H29NO13	575.16	593.19	575.16	0.8	5.969
	17.69	Blestrianol C	Terpenoid	C37H30O7	586.19	609.01	586.19	-0.7	4.884
	16.01	Cynanoside G	Glycoside	C41H62O16	810.40	425.18	810.40	-1.6	5.330
	13.39	Mahuannin D	Terpenoids	C30H44O9	528.142	529.15	528.14	1.9	4.3833

UHPLC-MS/MS: Ultra-high performance liquid chromatography-tandem mass spectrometry.

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Figure 3: UHPLC-MS chromatogram of Ethyl acetate extract of leaf of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: Ultra high performance liquid chromatography-mass spectrometry.

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The ethyl acetate extract of the stem of C. mimosodes identified >750 compounds, with >55 peaks. Most compounds belong to the classes of glycosides, terpenoids, saponins, and polyphenols, with other compounds in minute quantities. Among these, phanginin I was detected in 10 peaks, and other important phytochemicals detected are cartomin, blestrianol C, cynanoside G, and mahuannin D [Table 2, Figure 4].

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Figure 4: UHPLC-MS chromatogram of the ethyl acetate extract of the stem of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: Ultra high performance liquid chromatography-mass spectrometry.

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C. Methanol extract: Leaf and stem

The methanol extract of the plant’s leaves detected >365 compounds, with seven peaks visible on the chromatogram. Polyphenols, glycosides, and alkaloids are a significant class of compounds that have been detected. 8-gingerol, chloranoside A, quindoline, and bufotalin were identified as essential phytochemicals [Table 3, Figure 5].

Table 3: The major compounds identified from the methanol extract of theleaf and stem of C. mimosoides according to UHPLC-MS/MS.

Leaf extract	tR (min)	Compound name	Classification	Formula	Calculated mass	m/z	Extract mass	Mass accuracy (mDa)	% Base peak
	26.25	(8)-Gingerol	Polyphenols	C19H30O4	322.21	173.10	322.214	-0.5	15.122
	16.04	Chloranoside A	Glycosides	C21H28O9	424.17	425.18	424.17	2	10.107
	14.61	Quindoline	Alkaloids	C15H10N2	218.08	140.01	218.08	1	2.9689
	5.43	Bufotalinin	Cardenolides	C24H30O6	414.20	453.16	414.20	-0.7	3.5818
	16.04	Bidenoside C	Saponins	C16H22O6	310.14	173.10	310.142	0.4	3.2374
	16.01	Schisanhenol	Phenylpropanoids	C23H30O6	402.20	425.19	402.20	-1.8	7.1574
Stem extract	2.74	Mulberrofuran C	Polyphenols	C34H28O9	580.17	291.09	580.17	-2.2	6.1944
	10.58	Rubricauloside	Saphonins	C27H38O15	602.22	313.10	602.22	2	5.7966
	20.32	Erucamide	Fatty amide	C22H43NO	337.33	338.34	337.33	-1.2	5.623
	5.88	Perlolyrine	Alkaloids	C16H12N2O2	264.08	303.05	264.09	1.2	4.575

UHPLC-MS: Ultra high performance liquid chromatography-mass spectrometry.

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Figure 5: UHPLC-MS chromatogram of the methanol extract of the leaf of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: Ultra-high performance liquid chromatography-tandem mass spectrometry.

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The methanol extract of the stem contained the maximum number of compounds, with >855 compounds and 76 peaks in the chromatogram. Polyphenols, alkaloids, followed by saponins, and phenylpropanoid lignin were the major classes of compounds detected. Among the lignans, schisanhenol was the primary compound, followed by mulberrofuran C, rubricauloside, erucamide, and perlolyrine [Table 3, Figure 6].

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Figure 6: UHPLC-MS chromatogram of the methanol extract of the stem of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: Ultra-high performance liquid chromatography-mass spectrometry.

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D. Aqueous extract: Leaf and stem

In our study, the aqueous extract of the plant leaves was found to contain >250 compounds that were identified. Among these alkaloids are the majority, followed by polyphenols of the flavonoids belonging to the flavanol group, terpenoids (sesquiterpenoids), and saponins.

Major phytochemicals detected include capsaicin, which was detected in approximately five peaks, sanggenol, 6,7-dihydroxy-3,7-dimethyloct-2-enoic acid, javanicolide D, and centellasaphonin B [Table 4, Figure 7].

Table 4: The major compounds identified from the aqueous extract of the leaf and stem of C. mimosoides according to UHPLC-MS/MS.

Leaf extract	tR (min)	Compound name	Classification	Formula	Calculated mass	m/z	Extract mass	Mass accuracy (mDa)	% Base peak
	26.25	Capsaicin	Alkaloids	C25H28O6	305.19	173.11	305.19	-1.1	24.27
	16.04	Sanggenol A	Polyphenols	C18H27NO3	424.18	425.19	424.18	1.1	14.70
	26.33	6,7-Dihydroxy-3,7-dimethyloct-2-enoic acid	Terpenoids	C10H18O4	202.12	140.02	202.11	-0.6	5.48
	14.61	Javanicolide D	Terpenoids	C28H38O12	566.23	301.15	566.23	-2.4	4.91
	5.43	Centellasaponin B	Saphonins	C42H68O16	828.45	432.25	828.44	-1.5	2.75
Stem extract	16.02	Schisanhenol	Phenylpropanoids	C23H30O6	402.20	425.19	402.20	0.8	30.19
	14.59	Bruceajavanin B	Glycoside	C33H48O6	540.34	301.15	540.34	0.7	10.63
	20.33	Erucamide	fatty amide	C22H43NO	337.33	338.34	337.33	0.7	8.30
	5.69	12 beta-Hydroxy cinobufagin	Glycoside	C26H34O7	458.23	497.2	458.23	1.4	3.80
	26.25	Phanginin F	Glycoside	C21H28O5	376.18	189.1	376.18	-0.3	2.44

UHPLC-MS/MS: Ultra-high performance liquid chromatography-tandem mass spectrometry.

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Figure 7: UHPLC-MS chromatogram of aqueous extract of the leaf of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: Ultra-high performance liquid chromatography-mass spectrometry.

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The aqueous extract of the plant stems detected >279 compounds, with ten distinct peaks. Among these, phenylpropanoids belonging to the lignin family were detected in significant quantities, followed by glycosides, fatty amides, and polyphenols of the nonflavonoid group.

Major phytochemicals detected are schisanhenol, bruceajavanin B, erucamide, 12 beta-hydroxy cinobufagin, and phanginin F [Table 4, Figure 8].

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Figure 8: UHPLC-MS chromatogram of aqueous extract of the stem of Caesalpinia mimoisoides in positive ion mode. UHPLC-MS: ultra-high performance liquid chromatography-mass spectrometry.

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DISCUSSION

The compounds detected in the hexane extract of the leaves, such as ruvoside cardiac glycoside, which is found abundantly in Thevetia nerifolia, have not been studied extensively; however, they show cytotoxic effects on a few human cancer cell lines^[14,15] and have been shown to produce anti-inflammatory activity.^[16] Bicyclic peptide RAXiX is a cytotoxic bicyclic peptide found to have anticancer activity.^[17] Celosin F, found mainly in the Celosia argentea, has been found to possess various biological activities, such as anti-inflammatory, antitumor, and hepatoprotective activity.^[18] Ergosterol peroxidase is a compound that possesses antimicrobial, anti-inflammatory, neuroprotective, and anticancer properties. It produces a cytotoxic effect by modulating the Nrf2 and NF-κB signalling pathways, demonstrating its impact on colon cancer cell lines.^[19] 5-Undecyl-1,3-benzodioxole is an antitumor, antibacterial, antifungal, antioxidant, insecticide, and herbicide agent. It has been shown to possess potent inhibitory action on HER-2 receptors.^[20]

Hexane extract of the stem also detected celosin F, along with octadecanal, which is known to possess antioxidant effects along with antimicrobial, antigenotoxic, anticancer, and neuromodulator effects^[21] and anthraquinone purpurin, which is known to possess antioxidant, anti-inflammatory, neuroprotection, and anticancer properties,^[22] ginkgolid J is known to have antioxidant, anti-inflammatory and neuroprotective actions^[23] and Atractyloside D, a mitochondrial uncoupler, inhibited mitochondrial oxidative phosphorylation. It possesses anticancer activity by targeting fibroblasts and inhibiting metastasis. It is also found to be neuroprotective.^[24]

According to previous literature, quercetin was the major component detected in the aerial part of the plant in ethyl acetate extract. In contrast, quercetin, ethyl gallate, and gallic acid were detected in the entire plant ethanol extract. This finding differs from our study, where the primary compound detected was very different.^[25]

Among the phytochemicals from the ethyl acetate extract of the leaves, 3-phenylpyridine is the major component of the phenylpyridine group and exhibits significant antitumor, antimicrobial, antioxidant, and herbicidal properties.^[26] Cyanoside G, a pregnane glycoside isolated from Cyanachu auriculatum/atratum, possesses many biological activities, such as antitumor, neuroprotective, and anti-inflammatory activity. It has also been found to have an antifungal effect and is used in treating alzheimer’s disease.^[27] Caffeic acid, a phenolic acid, exhibits various biological properties, including antioxidant, anti-inflammatory, anticancer, and neuroprotective activities. Its antitumor activity has been studied in detail with multiple cell lines, establishing the mechanism of action, such as on cervical cancer lines (by inhibiting Bcl-2 activity), gastric cell lines (By alteration in cancer cell homeostasis and ERK1/K2 target), growth arrest and apoptosis in colonic cell lines via beta-catenin/T-cell factor signalling. The oxidative mechanism in the fibrosarcoma cell line prevents oxidative stress via the Nrf-2 pathway.^[28] Abrusoside A, another molecule extracted from Abrus precatorius, possesses antimicrobial, cytotoxic, anti-inflammatory, antioxidant, and antidiabetic activity. It has been found to modulate AMPK, MAPK, and Nrf2 signalling pathways.^[29] 2-Benzylidesuccinic acid, a derivative of succinic acid, exhibits biological activities through nuclear receptor ligand and enzyme inhibitors.^[30]

The ethyl acetate extract of the stem of C. mimosodes, identified as containing Phanginin I, which is also isolated from the seeds of Caesalpinia sappan, was found to have cytotoxic effects on various cell lines.^[31] Cartomin glycoside isolated was found to have various biological activities such as antioxidant, analgesic, hepatoprotective, and antidiabetic activity.^[32] Blestrianol C also exhibits a range of activities, such as antitumor, anti-inflammatory, antioxidant, and neuroprotective action. It has been demonstrated to exhibit antitumor activity against breast cancer cells through its antimitotic properties.^[33] Another phytochemical, Cynanoside G, detected in our study, was found to possess antiviral activity, anti-inflammatory activity, and immunosuppressive activity.^[34] Mahuannin D isolated was also found to possess antitumor and anti-inflammatory activity.^[35]

Analysis of methanol extract of the leaves detected 8-Gingerol as one of the compound found abundantly which is known to possess different biological activities like anti-inflammatory, antioxidant, and anticancer properties have been demonstrated in various cancer cell lines like colorectal cancer cell lines (regulate cancer cell proliferation through the EGFR/STAT/ERK pathway) and in the melanoma (by down regulating MAPK/PKA signal pathways).^[36] Chloranoside A has been found to possess multiple actions, including neuroprotective, antimicrobial, antioxidant, and cytotoxic effects.^[37] Bufotalinin, a cardiac glycoside found in toad skin and certain plants, is known to possess antiviral and anti-inflammatory activity along with proven anticancer activity via induction of apoptosis, G2/M phase disruptions, regulation of ROS, and BMT. It has shown its effect in triple-negative breast cancer by inhibiting the STAT3/EMT axis.^[38] Bidenoside C is found only in a few plants. The detailed impact of this phytochemical on biological function requires further elucidation.

The findings of our study were in contrast to those in the literature, where the phytochemical composition of the methanolic extract of leaves, as analysed using HPLC, showed the presence of gallic acid, catechin, caffeine, vanillic acid, ferulic acid, rutin, resveratrol, and quercetin as standard compounds.^[13,19] Another study revealed that the major phenolic compounds were gallic acid and ethyl gallate, with peak areas of 47.23% and 35.52%, respectively.^[39]

The methanol extract of the stem detected Schisanhenol as the primary compound, similar to the aqueous extract of the plant, which possesses hepatoprotective, antiviral, and potent anti-inflammatory properties. Also used in the treatment of cytokine syndrome in herbal medicine. It is known to induce apoptosis by suppressing the ASK1-P38-NF-κB pathway. It also attenuates endothelial dysfunction in cancer conditions.^[40-43] Mulberrofuran C, another active component isolated, is found to possess hepatoprotective, neuroprotective, anti-inflammatory, antineoplastic, and cardioprotective action.^[44] Erucamide, a bioactive fatty acid amide, possesses various activities like antidepressant, anxiolytic, antibacterial action, antiplasmodial, and anti-toxoplasma effects. It exhibits significant antitumor activity against lung cancer cell lines and other cancer cells, demonstrating a synergistic effect.^[45-48] Rubricauloside, even though isolated from the root of Umbelliferae, has not been studied in detail.^[49] Perlolyrine is primarily found in food items such as tomatoes and other compounds, exhibiting antiproliferative effects and possessing antidepressant and neuroprotective properties.^[50]

In the aqueous extract of the leaves, capsaicin, detected in around five peaks, is a significant component with potential analgesic, anti-inflammatory, antioxidant, and antimicrobial effects. Along with these, it also exhibits cardioprotective and metabolic activity and is a potential anticancer agent.^[51] Capsaicin is a potential antitumor compound in a wide variety of cancer types, including breast cancer, lung cancer, gastric cancer, liver cancer, prostate cancer, bladder cancer, and so on, through various mechanisms targeting pro-apoptotic genes and signalling pathways.^[52] other components are Sanggenol, Polyphenol compound found abundantly possesses Anti-inflammatory, Neuroprotective factor, and anticancer activity through the P53 inhibition and activation of caspase cascade in skin cancer like melanoma, inhibition of NF-KB signalling in ovarian cancer cell line and alteration in miR-26a-1-3p/MDM2/p53 signalling pathway,^[53,54] 6,7-Dihydroxy-3,7-dimethyloct-2-enoic acid (5%), exhibits antimicrobial, antioxidant, anti-inflammatory, and antitumor activity.^[55]

Javanicolide D has anti-inflammatory, antiviral, antitumor, and antidysentery activity. Its antitumor property has already been studied in various carcinomas, such as hepatocellular carcinoma and ovarian, breast, and cervical cancer. It has been shown to target different pathways in different cancers; in lung cancer, it has demonstrated its activity through targeting the JNK pathway, and in lymphocytic leukemia by inhibition of the PI3K/Akt pathway and down-regulating the c-myc gene.^[56]

In the aqueous extract of the plant stems, schisanhenol was the primary compound detected, similar to the methanol stem extract, followed by bruceajavanin B, which possesses anti-inflammatory, antibacterial, antimalarial properties, and anticancer activity in many cancer cell lines by inhibiting proliferation, apoptosis, migration, and invasion.^[57] Other phytochemicals, 2-Benzylidenesuccinic acid, are also found in the ethyl acetate extract of the leaf and have been shown to possess various biological activities. Erucamide is also detected in the methanolic extract of the stem.

CONCLUSION

The phytochemical analysis of C. mimosoides of the hexane extract of leaf and stem yielded triterpenoid saponins, sterols, polyphenols, and bicyclic hexapeptides as the major class. Ethyl acetate extract of leaves and stems yielded different phytochemicals, while leaf extracts yielded alkaloids, glycosides, non-flavonoid polyphenols, triterpenoids, and steroids, whereas stem extract was composed of glycosides, terpenoids, saponins, and polyphenols. Methanolic extracts of the leaf and stem showed similar results except for saponins and phenylpropanoid lignin in the stem. Aqueous extract of leaf and stem showed variations in predominant compounds; leaf yielded alkaloids, polyphenols of the flavonoids belonging to the flavanol group, terpenoids (sesquiterpenoids), and saponins, compared to stem, which yielded phenylpropanoids followed by glycosides, fatty amides, and polyphenols of the nonflavonoid group. We found in our study that the majority of the diverse chemicals were extracted with various solvents of the same part of the plant, and that the same solvent extraction of different parts of the plant also showed a majority of unique chemicals, which demonstrate that the plant has the potential to be used in the future to discover newer molecules for further therapeutic applications.