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Article

Quality Studies on Cynometra iripa Leaf and Bark as Herbal Medicines

by
Shabnam Sabiha
1,
Kamrul Hasan
1,
Katelene Lima
1,
Maryam Malmir
1,
Rita Serrano
1,
Isabel Moreira da Silva
1,
João Rocha
1,
Nurul Islam
2 and
Olga Silva
1,*
1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal
2
Department of Zoology, Faculty of Biological Sciences, University of Rajshahi, Rajshahi 6205, Bangladesh
*
Author to whom correspondence should be addressed.
Molecules 2024, 29(11), 2629; https://doi.org/10.3390/molecules29112629
Submission received: 12 April 2024 / Revised: 20 May 2024 / Accepted: 22 May 2024 / Published: 3 June 2024
(This article belongs to the Special Issue Extraction, Purification, Structural Analysis, Biological Evaluation, and Molecular Mechanisms of Dietary Bioactive Compounds from Natural Resources)
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Versions Notes

Abstract

:
Cynometra iripa Kostel. is a Fabaceae species of mangrove used in traditional Ayurvedic medicine for treating inflammatory conditions. The present study aims to establish monographic botanical and chemical quality criteria for C. iripa leaf and bark as herbal substances and to evaluate their in vitro antioxidant potential. Macroscopic and microscopic qualitative and quantitative analyses, chemical LC-UV/DAD-ESI/MS profiling, and the quantification of key chemical classes were performed. Antioxidant activity was evaluated by DPPH and FRAP assays. Macroscopically, the leaf is asymmetrical with an emarginated apex and cuneate base. Microscopically, it shows features such as two-layered adaxial palisade parenchyma, vascular bundles surrounded by 3–6 layers of sclerenchyma, prismatic calcium oxalate crystals (5.89 ± 1.32 μm) along the fibers, paracytic stomata only on the abaxial epidermis (stomatal index–20.15), and non-glandular trichomes only on petiolules. The microscopic features of the bark include a broad cortex with large lignified sclereids, prismatic calcium oxalate crystals (8.24 ± 1.57 μm), and secondary phloem with distinct 2–5 seriated medullary rays without crystals. Chemical profile analysis revealed that phenolic derivatives, mainly condensed tannins and flavonoids, are the main classes identified. A total of 22 marker compounds were tentatively identified in both plant parts. The major compounds identified in the leaf were quercetin-3-O-glucoside and taxifolin pentoside and in the bark were B-type dimeric proanthocyanidins and taxifolin 3-O-rhamnoside. The total phenolics content was higher in the leaf (1521 ± 4.71 mg GAE/g dry weight), while the total flavonoids and condensed tannins content were higher in the bark (82 ± 0.58 mg CE/g and 1021 ± 5.51 mg CCE/g dry weight, respectively). A total of 70% of the hydroethanolic extracts of leaf and bark showed higher antioxidant activity than the ascorbic acid and concentration-dependent scavenging activity in the DPPH assay (IC50 23.95 ± 0.93 and 23.63 ± 1.37 µg/mL, respectively). A positive and statistically significant (p < 0.05) correlation between the phenol content and antioxidant activity was found. The results obtained will provide important clues for the quality control criteria of C. iripa leaf and bark, as well as for the knowledge of their pharmacological potential as possible anti-inflammatory agents with antioxidant activity.

Graphical Abstract

1. Introduction

Cynometra iripa Kostel., commonly known as “Shingra”, is classified as “Least Concern” in the IUCN Red List of Threatened Species [1]. It is a globally recognized mangrove species [2,3] that belongs to the Fabaceae (Leguminosae) family and to the polyphyletic Cynometra L. genus, which comprises 113 species of shrubs to large trees [4,5,6,7,8,9,10]. C. iripa is a characteristic species of mangrove swamps [11], exhibiting a scattered distribution. It is present in various regions, including India, Bangladesh, Myanmar, Thailand, Northeast Australia, Papua New Guinea, Eastern Indonesia (such as West Irian, Halmahera, Moluccas, Seram, Ambon, Aru, and Tanimbar Islands), and the Philippines, ranging from Panay Island to Mindanao [1].
C. iripa is a small tree (6–15 m), sometimes multi-stemmed (Figure 1). The leaf (Figure 1a) is green color, 1–2-jugate, and asymmetrical. The flower is aromatic and appears in white or a delicate pale pink hue. The fruit is one-seeded, asymmetrical, with a pronounced beak at the apex of the dorsal suture, extending partially along the dorsal side. It is suborbicular, laterally compressed, deeply wrinkled, woody, and transitions from green to brown as it matures. The bark (Figure 1b) is smooth, displays brown-grey and patchy tones, and is finely fissured [12,13,14].
Traditionally, in India, a paste of the leaf, seed, and stem of C. iripa is used to heal wounds [15], and a decoction of the leaf is used to treat ulcers [16]. Tribal people extract oil from the seeds to treat cholera [17]. Although this species is found in Bangladesh and traditionally used by local people, no specific therapeutic indications have been found in the literature.
Some chemical studies have already been conducted on different C. iripa plant parts (Table 1). A total of 10 fatty acids were detected in the leaf oil, while 14 fatty acids were detected in the seed oil [18]. Fifteen compounds were identified by GC-MS from the seed and seed coats of this species [19,20]. Basak et al. (1996) reported the presence of chlorophyll, carotenoids, proteins, polyphenols, and tannins in the leaf ethanolic extract of this species. The carotenoid, polyphenol, tannin, and protein contents were 0.08, 30.15, 18.34, and 22.58% of the dry weight of the extract, respectively [21].
Methanol, ethyl acetate, and chloroform–methanol (1:1) extracts of C. iripa leaf showed antibacterial activity against two strains of Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Vibrio alginolyticus by the diffusion method [22]. It has also been reported that an ethanolic and methanolic extract of the C. iripa aerial parts showed in vitro antimicrobial activity against Bacillus cereus, P. aeruginosa, Staphylococcus aureus, and Salmonella typhimurium through the diffusion method. In comparison to ethanolic extracts, the methanolic extracts of leaf, stem, early seed, mature seed, and seed coat presented higher antimicrobial activity against P. aerunginosa [23]. In addition, the methanol extract of the bark showed antifungal activity against Alternaria alternata and Fusarium moniliforme using the poison food technique [24].
C. iripa is often confused with another species, Cynometra ramiflora (L.), and previously, it was described as a variety of this species. According to the original description made by Linnaeus (1753), C. ramiflora is characterized by unijugate leaves [25], whereas Kosteletzky (1835) described C. iripa as having bijugate leaf [26]. For this reason, C. ramiflora var. bijuga has been considered a synonym of C. iripa [11]. Although based on key characteristics like the apex of leaflets, the length of inflorescences, the length of pedicels, the apex of the anthers shape, and the position of the fruit beak, this species is considered to be different [12].
As different plant parts of this species are used in Ayurveda and other systems of Indian traditional medicine, to allow their use as herbal medicines, monographic quality parameters are essential, which is the main goal of the present work. Concerning the identification, macroscopic and microscopic analyses of the whole, fragmented, and powdered plant materials (leaf and bark) are performed together with the establishment of the chemical fingerprints and quantification of the main class of compounds. Additionally, in attending to the chemical fingerprint results, the antioxidant potential of the extracts of these medicinal plants is also evaluated.

2. Results

2.1. Macroscopic and Microscopic Analyses

2.1.1. Leaf

Macroscopic Characteristics

The macroscopic observation (Figure 2 and Figure 3) revealed a dried grey color asymmetrical leaflet, with leaf lamina 3.0–6.6 cm in length and 1.4–2.8 cm wide, emarginate apex (Figure 3a) and cuneate base (Figure 3c), and venation brochidodromous, prominent on the abaxial surface (Figure 3b) and entire margin. Trichomes were observed on leaf rachis and petiolules (0.2–0.3 cm long) (Figure 3d).

Microscopic Characteristics

Light microscopy (LM) analysis of transversal sections of C. iripa leaf showed rectangular to polygonal upper epidermis cells, smaller (10.26–18.64 µm) than the lower epidermis cells (12.84–34.20 µm), mucilage-containing cells on the abaxial epidermis, and double-layered palisade parenchyma on the adaxial mesophyll tissue. Hypodermal layers were absent. The presence of adaxial xylem and abaxial phloem surrounded by 3–6 layers of sclerenchyma was observed (Figure 4a,b).
The paracytic type of stomata (two subsidiary cells parallel to the guard cells) was only detected on the abaxial surface (Figure 4c,d). Calcium oxalate prismatic crystals (3.52–8.70 µm) were observed in the veins (Figure 4e), and unicellular and pointed non-glandular trichomes were observed in the petiolules (Figure 4f).
The leaf powder of C. iripa was greyish-green in color and had a specific odor. By LM, it was possible to identify the presence of characteristic leaf microscopic elements like palisade parenchyma consisting of two layers of cells, fibers, free calcium oxalate prismatic crystals, and free non-glandular trichomes (Figure 5a–d).

2.1.2. Bark

Macroscopic Characteristics

The dried stem bark was nearly flat in the piece, smooth, brown-grey in color, and finely fissured; thickness is usually 2–3 mm (Figure 6a,b).

Microscopic Characteristics

LM analysis of the C. iripa bark transversal sections showed the presence of lenticel, periderm, narrow phelloderm (composed of tangentially elongated cells), broad cortex with large elliptical groups of sclereids (heterogenous in shape and size), parenchyma (Figure 7a,b), numerous calcium oxalate prismatic crystals (Figure 7c), and secondary phloem with distinct 2–5 seriated medullary rays (Figure 7d). No calcium oxalate prismatic crystals were found on medullary rays (Figure 7d).
The LM longitudinal section analysis revealed the presence of fibers, some with calcium oxalate prismatic crystals along and parenchyma cell layers and numerous irregularly shaped starch granules (1.65–5.96 µm), isolated or conjugated, scattered in cell layers (Figure 7e,f).
The powdered C. iripa bark was greyish brown in color and characterized by the presence of fragments of fibers (Figure 8a), fragments of parenchyma and reddish-brown periderm (Figure 8b), calcium oxalate prismatic crystals (Figure 8c), and occasional starch granules.

2.2. Quantitative Microscopic Analysis

The principal microscopical characteristics of C. iripa leaf and C. iripa bark were quantified to provide additional distinctive elements for quality control purposes of these medicinal plants as possible herbal drugs. The results are presented in Table 2. Noticeably, the abaxial epidermal cells were larger in the leaf than the adaxial epidermis cells, and the calcium oxalate prismatic crystals were wider in the bark than in the leaf.

2.3. Chemical Studies

2.3.1. Yield of Extraction

Chemical studies were performed using extracts prepared with botanically characterized raw plant materials. The obtained extraction yields and drug extract ratio (DER) are presented in Table 3. The extract yield percentage was higher in C. iripa bark (CIB) than in the C. iripa leaf (CIL), corresponding to a lower DER, as verified.

2.3.2. Qualitative Phytochemical Analysis

A portion of each extract (CIL and CIB) was analyzed using characteristic colorimetric methods for secondary metabolites. The results (Table 4) confirm the absence of alkaloids in both plant extracts, whereas the presence of phenolic compounds (in ferric chloride test, the bluish-black color formation, and in the acetic acid test, the red color formation) and triterpenoids, namely saponins (stable foam formation), is confirmed in both (CIL and CIB) extracts.

2.3.3. LC-UV/DAD-ESI/MS Fingerprint

The obtained results of the analysis by high-resolution liquid chromatography coupled to a photodiode array and a mass spectrometry detector using electrospray ionization (LC-UV/DAD-ESI/MS) are presented in Table 5 and Table 6 and Figure 9. The tentative identification of the main compounds was assigned by co-chromatography with authentic standards, comparison of their UV spectra and retention time, and mass spectrometric data based on the PubChem database and different scientific literature. Negative ionization data were selected for identification.
Table 5 presents data on the main compounds identified from C. iripa leaf extracts by LC-UV/DAD-ESI/MS. The obtained chromatograms for CIL extracts showed a total of 11 major peaks. Peak a showed a [M − H]− ion at m/z 1154 and fragment ions at m/z 865 [M − H − 289]−, 577 [M − H − 289 − 288]−, and 425 [M − H − 289 − 288 − 152 ]− that formed due to RDA fragmentation (one of the most common fragmentation pathways of B-type proanthocyanidins), and 287, that is a monomeric catechin unit, formed due to quinone methide cleavage [27]. Notably, for B-type proanthocyanidins, fragments form the monomeric ions of m/z 287 or m/z 289 [27], and according to Karonen et al., 2004, B-type procyanidin oligomers are composed of multiple monomer subunits with interflavonoid C-C linkages that differ by multiples of 288 [28]. Considering the differences between monomer units, UV spectra, and fragmentation pattern, this compound was identified as a B-type proanthocyanidins tetramer.
Peak b exhibited a [M − H]− ion at m/z 865 and fragment ions at m/z 577 [M − H − 288]−, and 289 (monomeric catechin unit) was identified as a B-type proanthocyanidins trimer. Peaks c and d showed the [M − H]− ion at m/z 1442 and subsequent fragment ions at m/z 1154 [M − H − 288]−, 865 [M − H − 288 − 289]−, 577 [M − H − 288 − 289 − 288]−, and 289 (monomeric catechin unit), which indicated a molecular weight of 1443. Based on the differences between monomer units, UV spectra, and fragmentation pattern, these compounds were also identified as B-type proanthocyanidin pentamers.
Peaks e, f, and h presented a [M − H]− ion at m/z 435, corresponding to a molecular weight of 436, and produced taxifolin aglycone fragment ions at m/z 303 [M − H − 132]− (indicating loss of a pentose moiety), 285 [M − H − 132 − 18]− (indicating loss of water), and 151 (a fragment produced due to a RDA reaction). The pentose moiety could be attributed to arabinose or xylose. Since arabinose and xylose are monosaccharides with the same molecular formula (C5 H10 O5) and molecular weight (150 g/mol), more experiments are needed to obtain the correct identity of the sugar moiety in these peaks and based on the UV–Vis and MS spectral data, these peaks have tentatively been identified as taxifolin pentoside isomers [29,30].
Peak g showed a [M − H]− ion at m/z 463 corresponding to molecular weight 464, with respective fragment ions at m/z 435 [M − H − 28]−, indicating loss of CO, and 301 [M − H − 162]−, indicating loss of a glycosyl unit. Based on the UV, fragmentation pattern, and co-chromatography with standards (quercetin-3-O-glucoside), this compound was assigned as quercetin-3-O-glucoside [31,32].
Peak i showed [M − H]− ion at m/z 433 and characteristic fragment ion at m/z 301 [M − H − 132]−. By comparison of its fragmentation behavior with previous work in the literature, this peak was tentatively identified as quercetin 7-O-pentose/apiose [33].
Peak j showed an m/z 447 [M − H]− and fragment ions at m/z 419 [M − H − 28]−, indicating loss of CO, and 285 [M − H − 162]−, a Kaempferol aglycone formed by the loss of the glycosyl unit and a UV spectrum compatible with its flavonol nature, namely kaempferol-7-O-glucoside (MW 448 g/mol). This identity was confirmed by the spectral information of the kaempferol 7-O-glucoside of the PubChem database [34].
Peak k showed a base peak at m/z 269 [M − H]− with fragment ions at m/z 89 and a UV spectrum compatible with its flavone nature, namely apigenin (MW 270 g/mol). This identity was also confirmed by LC/UV-DAD co-chromatography with authentic standards (apigenin).
Table 6 presents data on the main compounds identified from C. iripa bark extracts by LC-UV/DAD-ESI/MS. The obtained chromatograms for CIB extracts showed a total of 11 major peaks. Both peaks a′ and c′ showed a [M − H]− ion at m/z 1154 and a similar fragmentation pattern corresponding to the B-type proanthocyanidins tetramer that was observed for peak a in CIL. Peak b′ also showed a [M − H]− ion at m/z at 865 with the fragmentation behavior of the B-type proanthocyanidins trimer, which was also similar to peak b in CIL.
Peak d′ and e′ exhibited a [M − H]− ion at m/z at 561 and fragment ion at m/z 433 [M-H-126]− which formed due to RDA fragmentation. The other two fragment ions 287 and 273/271 formed due to a quinine methide reaction (QM) that indicated catechin and afzelechin derivatives, respectively. Based on the UV and fragmentation patterns reported in previous work, these peaks were identified as B-type proanthocyanidins dimers [35].
Peak f′ showed a [M − H]− ion at m/z 565 and fragment ion 301 [M − H − 264]−, a typical fragment for quercetin derivatives formed by the loss of two pentose units (132 + 132). Concerning the UV and fragments behavior, this compound was tentatively identified as quercetin-3-O-pentosyl-pentoside [36,37].
Peak g′, h′, and i′ presented a [M − H]− ion at m/z 449 and characteristic fragment ions at m/z 303 [M − H − 146]− assigned to [aglycone H]− 285 and 151, which is similar fragmentation behavior to taxifolin. Based on the obtained UV–Vis and MS spectral data, this peak was tentatively identified as a deoxyhexose (rhamnose) of taxifolin, namely as taxifolin 3-O-rhamnoside [38].
Peak j′ exhibited a [M − H]− ion at m/z 599 and fragment ions at m/z 447 [M − H − 152]− formed by the removal of the galloyl moiety and the other fragment ion at m/z 301 [M − H − 298 (152 + 146)]−, indicating the removal of the galloyl-rhamnoside moiety. Based on the UV and typical fragmentation behavior, this compound was identified as quercitrin 3″-O-gallate [39].
Peak k′ was tentatively identified as apigenin, similar to peak k in CIL as it exhibited a base peak at m/z 269 [M − H]− with fragment ions at m/z 89 corresponding to a molecular weight of 270.
Quercetin-3-O-glucoside (Figure 10a) and taxifolin pentoside (Figure 10b) were found as the major compounds identified in the CIL, whereas B-type dimeric proanthocyanidins (Figure 10c) and taxifolin 3-O-rhamnoside (Figure 10d) were the main compounds identified in the CIB extracts.

2.3.4. Quantitative Phytochemical Analysis

From the qualitative analysis, phenolic derivatives were identified as the main chemical class. For this reason, the total phenolic content (TPC), total flavonoid content (TFC), and total condensed tannin content (TCTC) were determined in both extracts and are presented in Table 7. Gallic acid, catechin, and cyanidin chloride were used as standard, respectively.
TPC was significantly higher (p < 0.05) in CIL than CIB, whereas TFC and TCTC were higher (p < 0.05) in the CIB extract.

2.4. Antioxidant Activity

2.4.1. DPPH Scavenging Activity

The results of the scavenging activities of the CIL and CIB extracts by the DPPH method are presented in Figure 11. Both extracts showed a higher antioxidant activity than ascorbic acid (ASC) and concentration-dependent scavenging activity.
In fact, the IC50 (half maximal inhibitory concentration) values of CIL and CIB are similar (23.95 ± 0.93 and 23.63 ± 1.37 µg/mL, respectively) and lower than the obtained value of ASC (30.75 ± 0.51 µg/mL), indicating the higher antioxidant activity of the extracts in comparison with this recognized antioxidant. However, the CIB extract showed the highest percentage of scavenging, 80.3%, at a concentration of 40 µg/mL.

2.4.2. Ferric Reducing Capability

The results of the ferric-reducing capacity of CIL and CIB determined by the FRAP test are presented in Figure 12. Both the CIL and CIB extracts showed a lower ferric reduction capacity than quercetin and ascorbic acid used as standards. The FRAP value of the CIL extract is 61.11 ± 2.91 µmol Fe2+/g dry weight, and of the CIB extract, this is 77.94 ± 2.02 µmol Fe2+/g dry weight, while the FRAP value of the quercetin and ASC was 121.51 ± 0.94 µmol Fe2+/g dry weight and 149.84 ± 1.08 µmol Fe2+/g dry weight, respectively. Comparatively, the CIB extract was shown to have more antioxidant potential than CIL.

2.5. Correlation between Phenolic Content and Antioxidant Activity

The results of the statistical calculation concerning the possible correlation between the phenolic content of the CIL and CIB extracts and the antioxidant potential are presented in Table 8. For both the CIL and CIB extracts, a positive and statistically significant correlation (p < 0.05) has been found between the phenolic content and antioxidant activity.
For both the CIL and CIB extracts, TFC and TCTC showed a positive and strong correlation with FRAP activity, giving a Pearson correlation coefficient (r) of 0.99. On the other hand, TPC was positively correlated with DPPH activity, giving an r of 0.85 and 1.00 for CIL and CIB, respectively (Table 8).
Therefore, the results indicate that different phenolic derivatives, mainly procyanidins, made an outstanding contribution to the antioxidant activity of the CIL and CIB extracts.

3. Discussion

The quality control of medicinal plant materials is essential to allow them to be used as herbal medicines for human and veterinary use [40]. Therefore, the botanical macroscopic and microscopic characteristics are essential for identifying whole, fragmented, and powdered samples of C. iripa leaf and C. iripa bark.
Considering the external leaf morphology observed, C. iripa dried leaf has features like alternate jugate leaf arrangement, emarginate apex, cuneate base, and petiolules size up to 0.3 cm similar to those reported by Cooper, W.E. (2015) [13] and Ragavan et al. (2017) [12] for the fresh leaf of this species. In addition, common Leguminosae features are noticed for the first time in this species, like paracytic stomata and calcium oxalate prismatic crystals. Saenger and West (2016) referred to the presence of a single palisade layer and the absence of the hypodermal layer as characteristics of C. iripa leaf [41]. Our results differ from their study, except for the hypodermal layer, as we found a double layer of palisade parenchyma on the adaxial. Other characteristics found in C. iripa leaf are the presence of a vascular bundle surrounded by layers of sclerenchyma, mucilage-filled cells, and paracytic stomata found only on the abaxial surface. Pan (2010) reported the presence of paracytic stomata only on the abaxial surface in another species, Cynometra chaka. Furthermore, in C. chaka and Cynometra lujae De Wild., multicellular uniseriate trichomes were found on the abaxial surface of the leaf [42], whereas in C. iripa, non-glandular unicellular trichomes were found only in the petiolule.
In this study, for the first time, the microscopic features of the dried bark of C. iripa are discussed. The most distinctive elements for quality control proposed are the presence of lenticel, periderm, narrow phelloderm, broad cortex with large elliptical groups of sclereids, calcium oxalate prismatic crystals, secondary phloem with 2–5 seriated medullary rays, and irregularly shaped starch granules in all parenchymatous tissues.
The chemical profile analysis showed that phenolic compounds, mainly condensed tannins, and flavonoids, are the main classes identified in C. iripa leaf and bark extracts. The major compounds identified in the leaf were quercetin-3-O-glucoside and taxifolin pentoside.
Quercetin and its glycosides are vital plant flavonoids with neuroprotective, cardioprotective, chemo-preventive, antioxidant, anti-inflammatory, and anti-allergic properties [43,44]. These compounds have been shown to suppress inflammatory responses by inhibiting inflammatory enzymes cyclooxygenase (COX) and lipoxygenase [44], and also by inhibiting the production of pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β), in various cell types [45,46]. Quercetin-3-O-glucoside demonstrated strong antioxidant and anti-inflammatory properties in vitro, as it showed the highest activity against cyclooxygenase (COX)-1, COX-2, and lipoxygenase (LOX-5), with IC50 values of 3.62, 5.66, and 2.31 µg/mL, respectively. Additionally, it exhibited considerable cytotoxic effects on HeLa cells in a dose- and time-dependent manner [47].
Taxifolin is a potent antioxidant that inhibits the increased activity of NF-κB in rats with cerebral ischemia–reperfusion injury [48]. It also exhibited notable anti-inflammatory effects by reducing the transcription of TNF-α, IFN-γ, IL-10, and TLR-4 in Raw 264.7 cells in mice [49]. The other marker compounds detected were B-type trimeric, tetrameric, and pentameric proanthocyanidins, quercetin 7-O-pentoside/apioside, kaempferol 7-O-glucoside, and apigenin. All these compounds are identified for the first time in C. iripa leaf. Different phenolic derivatives are reported to be found in other species of Cynometra. For example, proanthocyanidins, taxifolin pentoside, taxifolin 3-O-arabinofuranoside, catechin, apigenin 8-C-glucoside (vitexin), apigenin 6-C-glucoside (isovitexin), kaempferol hexoside, quercetin pentoside, quercetin hexoside, kaempferol–coumaroyl hexoside, isorhamnetin hexoside, and acacetin 7-O-β-glucoside have been isolated from the ethyl acetate and n-butanol, fractions of the leaf of Cynometra cauliflora L. [29,50].
The major compounds detected in the C. iripa bark were B-type dimeric proanthocyanidins and taxifolin 3-O-rhamnoside. Proanthocyanidins (condensed tannins) are reported to have significant antioxidant, anti-cancer, anti-diabetic, antimicrobial, and immunomodulatory potential [51]. Several studies in the literature reported on the different biological activities of B-type proanthocyanidins like anti-cancer activity by decreasing the in vitro growth of androgen-sensitive (LnCaP) and androgen-resistant (DU145) human prostate cancer cell lines [52], antimicrobial activity against Candida albicans and Cryptococcus neoformans, with MIC values of 250 to 1000 µg/mL [53], and anti-aging activity by reducing the content of ROS and nicotinamide adenine dinucleotide phosphate oxidases 4 (NOX4) mRNA levels in luteinized granulosa cells (hGC) and tumor granulosa cells (KGN) [54].
Taxifolin 3-O-rhamnoside is an important flavonoid that showed anti-tumor activity on PANC-1 and A-549 cancer cell lines by inhibiting about 30% of the cell growth at 30 µM concentrations [55]. The other marker compounds detected were B-type trimeric and tetrameric proanthocyanidins, quercetin 3-O-pentosyl-pentoside, taxifolin 3-O-rhamnoside, quercitrin 3″-O-gallate, and apigenin. Like C. iripa leaf, all these compounds are identified for the first time in the C. iripa bark. There are no other studies in the literature found concerning compounds in the C. iripa bark.
The bark extracts of different Fabaceae species, like Stryphnodendron adstringens (Mart.), Mimosa tenuiflora (Mart.), Mimosa arenosa (Willd.) Poir., Mimosa caesalpiniifolia Benth., Anadenanthera colubrina var. cebil. [56], and Plathymenia reticulata Benth [57], are a potential source of condensed tannins. Besides this, different flavonoids have been detected in the bark of different Fabaceae species like quercetin, quercitrin, taxifolin, apigenin, astibilin, and kaempferol, which have been identified in the Hymenaea martiana bark [58]. Another study reported the presence of isoquercitrin, quercetin, and rutin in Dimorphandra gardneriana Tul. bark [59].
However, the biological properties of polyphenols depend on their bioavailability [60,61] for intestinal absorption, metabolization, and subsequent interaction with target tissues or organs [62]. In fact, the metabolism of flavonoid glycosides involves several enzymatic activities and interactions with the gut microbiota, leading to the release of bioactive aglycones [63]. For instance, glycosylation improves the solubility and bioavailability of quercetin, which can enhance its therapeutic potential as quercetin has relatively low bioavailability due to poor absorption, rapid metabolism, and extensive first-pass elimination in the liver [64].
Imidazole alkaloids have been noticed in different plant parts of some Cynometra species, like anantine, cynometrine, and cynodine, from Cynometra anata Hutch. and Dalziel (leaf) [65], N1-demethyl cynometrine, N1-demethyl cynodine, cynometrine, and cynodine from Cynometra hankei Harms (stem bark and seed) [66] and anantine, cynometrine, isoanantine, isocynometrine, isocynodine, noranantine, hydroxyanantine, and cynolujine from C. lujae (plant part not referred) [65]. However, in our study, no trace of alkaloids was detected in C. iripa leaf (CIL) or C. iripa bark extracts.
The C. iripa leaf extract showed a higher total phenolic content (TPC), 1521 ± 4.71 mg of GAE/g dry weight, than the C. iripa bark extracts, which was 1476 ± 4.09 mg GAE/g dry weight. A higher TPC was also reported in C. cauliflora, in which the TPC of an aqueous extract of young leaf was 1831.47 ± 1.03 mg GAE/g [67] and a lower TPC was found in a C. ramiflora stem methanolic extract (96.2 mg GAE/g dry weight) exhibiting the influence of extraction methods in the quantification of the secondary metabolites in different Cynometra species [68].
The obtained values for the total flavonoid content (TFC) of C. iripa leaf and bark extracts were 64 ± 1.00 CE/g dry weight and 82 ± 0.58 mg CE/g, respectively. In a study, an aqueous extract of C. cauliflora leaf exhibited a TFC of 33.63 ± 0.25 mg CE/g dry weight [67], and a high TFC of 166.4 mg QE/g was reported in a methanol extract of C. ramiflora stem [68]. However, in our study, C. iripa bark extracts exhibited a higher TFC than the leaf extracts. In a study, the hydroethanolic and hydromethanolic extracts of a Fabaceae species named Pongamia pinnata (L.) Pierre bark showed a higher TFC 2.28 ± 0.01 and 3.44 ± 0.04 g CE/100 g dry weight, respectively, than the leaf extract [69]. Besides this, a higher TFC was also reported in the methanolic extract of stem bark (902 ± 0.7 mg quercetin equivalents/g) than the root and leaf extract of Rhizophora mucronate, which is also a mangrove species [70].
The total condensed tannin content (TCTC) of C. iripa leaf and C. iripa bark were 755 ± 4.4 mg and 1021 ± 5.51 mg CCE/g dry weight, respectively. A lower TCTC of 80.4 mg GAE/g dry weight was reported in another species C. ramiflora stem methanolic extract because of the differences in the species, plant part, extraction solvent, and methodology. In addition, we expressed our result in CCE (Cyanidin Chloride Equivalent), whereas they expressed their result in Gallic Acid Equivalent (GAE) [68]. No more studies were found in other Cynometra species concerning the tannin content in cyanidine chloride equivalent.
CIL and CIB extracts showed antioxidant activity by the DPPH assay with IC50 23.95 ± 0.93 and 23.63 ± 1.37 µg/mL, respectively, and by the FRAP assay with values of 61.11 ± 2.91 and 77.94 ± 2.02 µmol Fe2+/g dry weight, respectively. In the DPPH assay, both extracts showed a concentration-dependent scavenging activity higher than standard ascorbic acid. By the FRAP assay, both extracts showed a ferric-reducing capability lower than the used standards. Phenolic compounds, including procyanidins, were believed to be involved in the demonstrated antioxidant activity of both extracts. Comparatively, in both DPPH and FRAP assays, C. iripa bark (CIB) was shown to possess a higher antioxidant activity than C. iripa leaf (CIL). No more information has been found concerning the antioxidant activity of CIL and CIB hydroethanolic extracts. Ethanolic extracts of the leaf of another species C. cauliflora exhibited remarkable antioxidant activity with an IC50 value of 2.88 ± 0.05 µg/mL than the standard quercetin in the DPPH assay [71]. Also, aqueous extracts of fruit of the same species showed potent antioxidant capacity in both DPPH with an IC50 value of 0.47 ± 0.03 g of dry weight/mL and a FRAP assay with reducing power of 25.07 ± 0.73 µmol Fe2+/g dry weight [72].
A positive and statistically significant (p < 0.05) correlation was noticed between the phenolic content and antioxidant activity. So, these phenolic derivatives are mainly responsible for the antioxidant activity of both extracts.

4. Materials and Methods

4.1. Plant Materials

The leaf and bark of C. iripa were collected in April 2019 from the Koromjol and Harbaria eco-tourism center (Figure 13) in the Chadpai range of the Sundarbans, Khulna District, Bangladesh. The identification of the collected samples was confirmed by Dr. Fahmida Khanam, director, Bangladesh National Herbarium, and the corresponding voucher samples were deposited in this herbarium with the voucher number DACB-47644. A copy of them was also kept in the Laboratory of Pharmacognosy (Department of Pharmacy, Pharmacology, and Health Technologies) Faculdade de Farmacia, Universidade de Lisboa, (FFUL), Portugal.
After identification, the plant’s raw material for laboratory studies was dried in the dark at room temperature (±22 °C) in Bangladesh and transferred to the Laboratory of Pharmacognosy at the FFUL.

4.2. Botanical Studies

4.2.1. Samples

To conduct the botanical analysis, fifty samples were randomly selected from the 250 g of collected raw material in accordance with the sampling guidelines set forth in the European Pharmacopoeia for herbal drugs. A representative portion of the total collected plant material was powdered using a mill and then mounted in a 60% chloral hydrate solution, following the procedures outlined in the European Pharmacopoeia [73].

4.2.2. Macroscopic Analysis

The macroscopic analysis was performed with the naked eye and an Olympus SZ61 stereo microscope (Switzerland) equipped with a Leica MC170 HD digital camera. Image capture and analysis were facilitated by the Leica Application Suite (LAS) Version 4.8.0 software (Switzerland).

4.2.3. Microscopic Analysis

Transverse sections (midrib, distal part of the blade, and petiolule) and tangential longitudinal sections (leaf surface) were cleared and mounted in a 60% chloral hydrate aqueous solution. Microscopic analysis of the prepared leaf sections and powdered plant material was carried out using an Olympus CX31 microscope fitted with a Leica MC170 HD digital camera, with imaging processed via the LAS Version 4.8.0 software (Switzerland).
For macroscopic feature determination, observations were made on 15 adult leaves. For microscopic measurements, 30 samples were analyzed (1 mm² per sample). The stomatal index (SI) was calculated using the following formula:
SI = (S × 100)/(S + E)
where (S) represents the number of stomata per unit area of the leaf and (E) the number of epidermal cells in the same area of the leaf [73].

4.3. Chemical Studies

Plant Extract Preparation

The hydroethanolic (70%) extracts of each herbal substance (leaf and bark) were prepared using ethanol and water in a ratio of 70:30 at room temperature by maceration (a minimum of 3 × 24 h each). This solvent mixture assures the extraction of polar and apolar secondary metabolites. After extraction and filtration using the G4 glass filter under vacuum, the solution was evaporated by a rotary evaporator (Buchi R-100, Flawil, Switzerland) at a temperature less than 40 °C and then put in the freezer (−20 °C) and finally lyophilized at −55 °C (Heto LyoLab-3000, Dietikon, Switzerland) [73]. The Drug Extract Ratio (the ratio of the amount of plant material to the amount of the obtained extract) was evaluated, and the following equation was used to calculate the percentage (%) of yield:
Yield of extraction (%, w/w) = Wt1/Wt2 × 100%
Wt1 and Wt2 represent the final weight of the dried extract and the primary weight of the leaf/bark powder [74].

4.4. Qualitative Phytochemical Analysis

The hydroethanolic extracts were qualitatively analyzed for different secondary metabolites using conventional procedures and LC/UV-DAD/ESI-MS analysis. Preliminary phytochemical screening was conducted for alkaloids by the Bouchardat/Mayer/Dragendorff test [75], phenolic compounds by the ferric chloride test and acetic acid test [76], and saponins by the foam test [77].

LC-UV/DAD-ESI/MS Analysis

A Waters Alliance 2695 high-performance liquid chromatography (HPLC) system with an autosampler and photodiode array detector (Waters PDA 2996) was used in conjunction with a MicroMass Quattro MicroTM API triple quadrupole tandem mass spectrometer (Waters, Drinagh, Ireland). The separation module, also from Waters, included a quaternary pump system, degasser, autosampler, and column oven. Chromatograms were captured over a wavelength range of 210–700 nm.
An electrospray ionization source (ESI) was operated in negative mode. Separation was carried out using a LiCrospher® 100 RP-18 column (5 µm, 250 × 4 mm, Merck, Darmstadt, Germany) maintained at 35 °C. The flow rate was set to 0.3 mL/min with an injection volume of 20 μL. The mobile phase comprised water containing 0.1% formic acid (Phase A) and acetonitrile (Phase B), with a total run time of 90 min. The gradient conditions were 5% Phase B at 0 min, 20% Phase B at 20 min, 50% Phase B at 60 min, and 100% Phase B at 90 min. The peaks were analyzed by MassLynx™ V4.1 software (Waters®, Drinagh, Ireland). The compounds were identified by co-chromatography and by comparison of retention time, UV, and mass spectral data with reference standards (quercetin-3-O-glucoside from Honeywell Fluka, Germany, and apigenin from Extrasynthese, Genay, France) or tentatively identified according to the literature and databases.

4.5. Quantitative Phytochemical Analysis

All values were obtained in 3 sets of experiments and evaluated in triplicate by spectrophotometry using a Hitachi U-2000 UV–Vis spectrophotometer (Tokyo, Japan).
  • Total phenolic content
The total phenolic content of each of the extracts was determined using the Folin–Ciocalteu assay [78], where 2 mL of Folin–Ciocalteu reagent (diluted with water 1:10 v/v) was mixed with 0.4 mL of extract and then 1.6 mL of anhydrous Na2CO3 (75 g/L) solution. After two hours, the absorbance was measured at 765 nm. The gallic acid was used to obtain a standard calibration curve, and distilled water was used as blank. Results were expressed as mg of gallic acid equivalents (GAE)/g dried plant materials. Data are presented as the mean ± standard deviation.
  • Total flavonoid content
The total flavonoid content of each extract was determined by using the aluminum chloride colorimetric assay by Oliveira et al. (2008) with some modifications [79]. To 0.5 mL of extract, 2 mL of distilled water and 150 µL of 5% NaNO2 were added, and the mixture was left to incubate for 5 min. After that, 150 µL of 10% AlCl3 was added and incubated for 6 min. Then, finally, 1 mL of 1M NaOH was added and incubated at 18 °C in the dark for 20 min. Absorbance was measured at 510 nm. An increasing catechin concentration was used to obtain a standard calibration curve. The results were expressed as mg of catechin equivalents (CE)/g dried plant materials. Data are presented as the mean ± standard deviation.
  • Total condensed tannin content
The total condensed tannin content of each of the extracts was evaluated using the method of Porter et al. (1986) [80]. To 0.5 mL of plant extracts (diluted in 70% Acetone) we added 3 mL of butanol–HCl reagent (butanol-HCI 95:5 v/v) and 0.1 mL of ferric reagent (2% ferric ammonium sulfate in 2N HCl). Then, the solution was mixed and incubated at 97 to 100 °C for 1 h using a hot water bath. Absorbance was measured at 550 nm. The cyanidin chloride concentration was used as the standard calibration curve. The blank for each sample comprised 0.5 mL of the extract, 3 mL of butanol–HCl reagent, and 0.1 mL of the ferric reagent. Results were expressed as mg of cyanidin chloride (CCE)/g dried plant material. Data are presented as the mean ± standard deviation.

4.6. Antioxidant Activity

4.6.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Free Radical Scavenging Assay

The free radical scavenging activity was determined by the DPPH assay [81]. In this assay, the purple-colored DPPH becomes reduced by a hydrogen or electron donor, and its color changes to yellow. DPPH solution (3.9 mL, 6 × 10−5 M in methanol) was mixed with 100 µL of each extract. After 30 min of incubation at room temperature, the absorbance of the samples and standard solution was measured at 517 nm. Ascorbic acid was used as the reference standard. The inhibition ratio (percent) was calculated from the following equation.
% Inhibition = A0 − A1/A0 × 100
where A0 = the absorbance of the control and A1 = the absorbance of the standard.
The IC50 value is the concentration of the sample required to scavenge 50% of free radicals, and we calculated this from the plot of % inhibition against the concentration of each extract.

4.6.2. FRAP Assay

Under acidic conditions, the ferric 2,4,6-tri-2-pyridyl-s-triazine (Fe³⁺-TPTZ) complex is reduced to its ferrous form (Fe²⁺) by antioxidants, resulting in a vivid blue coloration with an absorption peak at 593 nm [82,83].
To prepare the FRAP reagent, 25 mL of acetate buffer (pH 3.6), 2.5 mL of ferric chloride solution (prepared by dissolving 0.5406 g of ferric chloride in 100 mL of distilled water), and 2.5 mL of TPTZ solution (prepared by dissolving 0.0781 g of TPTZ in 40 mM of HCl) were combined. The mixture was then incubated in a water bath at 37 °C for 10 min. For the assay, 300 μL of water and 100 μL of the test sample were added to a cuvette. About 3000 μL of the prepared FRAP reagent was subsequently introduced into the cuvette and mixed by inversion. A control assay was performed using water in place of the sample. The absorbance at 593 nm was recorded with a spectrophotometer exactly 4 min after adding the FRAP reagent.

4.7. Statistical Analysis

All the macroscopic and microscopic results were obtained by using Excel 365 software (version 2401) from Microsoft and expressed as minimum, maximum, mean ± SD, and median, except for determining the stomatal index (Table 2). Pearson’s correlation test was used to establish the correlation between TPC, TFC, TCTC, and antioxidant assays (DPPH, FRAP).

5. Conclusions

C. iripa is a medicinally important mangrove species. The different plant parts of this species are traditionally used to treat different ailments in Bangladesh and India. There are a few studies concerning the quality, safety, and efficacy of Cynometra species. For the first time, this study has been conducted on the establishment of quality parameters for C. iripa leaf and C. iripa bark as herbal medicines. C. iripa extracts have been found to be a good source of phenolic derivatives, mainly proanthocyanidins, believed to be responsible for their antioxidant activity. However, our future step will be a deeper phytochemical investigation to identify more secondary metabolites and their isolation, and besides this, pharmacological studies will be conducted to clarify their traditional use.

Author Contributions

Conceptualization, S.S. and O.S.; methodology, S.S., K.H., K.L., M.M., R.S. and O.S.; software, S.S., K.H., K.L. and O.S.; validation, S.S., K.H., K.L. and O.S.; formal analysis, S.S., K.H., K.L., M.M. and J.R.; investigation, S.S., K.H., K.L., R.S. and O.S.; resources, O.S.; writing—original draft, S.S.; writing—review and editing, M.M., R.S., I.M.d.S., J.R., N.I. and O.S.; supervision, J.R. and O.S.; project administration, J.R. and O.S.; funding acquisition, J.R. and O.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Foundation for Science and Technology (FCT, Portugal) through national funds to iMed.ULisboa (UIDP/04138/2020).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors are thankful to the Bangladesh National Herbarium (DACB), Bangladesh, and acknowledge the financial support from Fundação para a Ciência e Tecnologia and Portugal 2020 to the Portuguese Mass Spectrometry Network (LISBOA-01-0145-FEDER-402-022125).

Conflicts of Interest

The authors confirm that the content of this article has no conflicts of interest.

References

  1. The IUCN Red List of Threatened Species. Cynometra iripa. 2023. Available online: https://www.iucnredlist.org/species/178829/7619971 (accessed on 5 January 2023).
  2. Duke, N.C. Mangrove floristics and biogeography. In Tropical Mangrove Ecosystems; Robertson, A.I., Alongi, D.M., Eds.; American Geophysical Union: Washington, DC, USA, 1992; pp. 63–100. [Google Scholar] [CrossRef]
  3. Polidoro, B.A.; Carpenter, K.E.; Collins, L.; Duke, N.C.; Ellison, A.M.; Ellison, J.C.; Farnsworth, E.J.; Fernando, E.S.; Kathiresan, K.; Koedam, N.E.; et al. The loss of species: Mangrove extinction risk and geographic areas of global concern. PLoS ONE 2010, 5, e10095. [Google Scholar] [CrossRef] [PubMed]
  4. Bruneau, A.; Forest, F.; Herendeen, P.S.; Klitgaard, B.B.; Lewis, G.P. Phylogenetic relationships in the Caesalpinioideae (Leguminosae) as inferred from chloroplast trnL intron sequences. Syst. Bot. 2001, 26, 487–514. [Google Scholar] [CrossRef]
  5. Bruneau, A.; Mercure, M.; Lewis, G.P.; Herendeen, P.S. Phylogenetic patterns and diversification in the Caesalpinioid legumes. Botany 2008, 86, 697–718. [Google Scholar] [CrossRef]
  6. Mackinder, B.A.; Saslis-Lagoudakis, H.; Wieringa, J.J.; Devey, D.; Forest, F.; Bruneau, A. The tropical African legume Scorodophloeus clade includes two undescribed Hymenostegia segregate genera and Micklethwaitia, a rare, monospecific genus from Mozambique. S. Afr. J. Bot. 2013, 89, 156–163. [Google Scholar] [CrossRef]
  7. Radosavljevic, A.; Herendeen, P.S.; Mackinder, B.A. Phylogeny of the detarioid legume genera Cynometra and Maniltoa (Leguminosae). Syst. Bot. 2017, 42, 670–679. [Google Scholar] [CrossRef]
  8. De la Estrella, M.; Forest, F.; Klitgård, B.; Lewis, G.P.; Mackinder, B.A.; de Queiroz, L.P.; Wieringa, J.J.; Bruneau, A. A new phylogeny-based tribal classification of subfamily Detarioideae, an early branching clade of florally diverse tropical arborescent legumes. Sci. Rep. 2018, 8, 6884. [Google Scholar] [CrossRef] [PubMed]
  9. POWO (Plants of the World Online). Facilitated by the Royal Botanic Gardens, Kew. 2017. Available online: https://powo.science.kew.org/ (accessed on 5 November 2021).
  10. LPWG (The Legume Phylogeny Working Group). A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny. Taxon 2017, 66, 44–77. [Google Scholar] [CrossRef]
  11. Meeuwen, M.S.K. A revision of four genera of the tribe Leguminosae–Caesalpinioideae–Cynometreae in Indomalesia and the Pacific. Blumea 1970, 18, 1–52. [Google Scholar]
  12. Ragavan, P.; Rana, T.S.; Ravichandran, K.; Jayaraj, R.S.C.; Sivakumar, K.; Saxena, A.; Mohan, P.M. Note on Identity and Distribution of Cynometra iripa Kostel and C. ramiflora L (Fabaceae) in the Andaman and Nicobar Islands, India. Check List 2017, 13, 805–812. [Google Scholar] [CrossRef]
  13. Cooper, W.E. A taxonomic revision of Cynometra L. (Fabaceae) in Australia with a new species from the wet tropics of Queensland and a range extension to the mainland. Austrobaileya 2015, 9, 393–403. [Google Scholar] [CrossRef]
  14. Flora and Fauna Web. Cynometra iripa Kostel, National Parks, National Parks Board, Singapore. 2023. Available online: https://www.nparks.gov.sg/florafaunaweb/flora/4/0/4007 (accessed on 5 January 2022).
  15. Sen, U.K.; Bhakat, R.K. Assessment of psammophytic medicinal plant diversity used among the rural communities in Coastal East Midnapore, West Bengal, India. J. Herbs Spices Med. Plants 2019, 26, 219–247. [Google Scholar] [CrossRef]
  16. Pattanaik, C.; Reddy, C.S.; Dhal, N.K.; Das, R. Utilisation of mangrove forests in Bhitarkanika wildlife sanctuary, Orissa. Indian J. Tradit. Knowl. 2008, 7, 598–603. [Google Scholar]
  17. Lalitha, P.; Sachithanandam, V.; Swarnakumar, N.S.; Sridhar, R. Review on anti-inflammatory properties of mangrove plants. Asian J. Pharm. Res. 2019, 9, 273–288. [Google Scholar] [CrossRef]
  18. Desai, M.N.; Chavan, N.S. Fatty acid profile of Cynometra iripa kostel. from seeds and leaves. Int. J. Pharm. Res. Dev. 2011, 2, 58–62. [Google Scholar]
  19. Desai, M.N.; Chavan, N.S. Bioactive compounds from seed and seed coat of Cynometra iripa a mangrove species. Univers. J. Pharm. 2014, 3, 1–5. Available online: https://www.researchgate.net/publication/270898502 (accessed on 5 January 2023).
  20. Sabiha, S.; Serrano, R.; Hasan, K.; Moreira da Silva, I.B.; Rocha, J.; Islam, N.; Silva, O. The Genus Cynometra: A review of ethnomedicine, chemical, and biological data. Plants 2022, 11, 3504. [Google Scholar] [CrossRef]
  21. Basak, U.C.; Das, A.B.; Das, P. Chlorophylls, carotenoids, proteins and secondary metabolites in leaves of 14 species of mangrove. Bull. Mar. Sci. 1996, 58, 654–659. [Google Scholar]
  22. Choudhury, S.; Sree, A.; Mukherjee, S.C.; Pattnaik, P.; Bapuji, M. In vitro antibacterial activity of extracts of selected marine algae and mangroves against fish pathogens. Asian Fish. Sci. 2005, 18, 285–294. [Google Scholar] [CrossRef]
  23. Desai, M.N.; Chavan, N.S. Antibacterial activity and phytochemical screening of Cynometra iripa kostel. Int. J. Pharma Bio Sci. 2010, 1, 1–4. [Google Scholar]
  24. Powar, P.S.; Chavan, S.R.; Gaikwad, D.K. Antifungal activity of mangrove bark. Int. J. Pharma Bio Sci. 2011, 2, 484–488. [Google Scholar]
  25. Linnaeus, C. Species Plantarum; Laurentius Salvius: Stockholm, Sweden, 1753; p. 382. [Google Scholar]
  26. Kosteletzky, V.F. Allgemeine Medizinisch-Pharmazeutische Flora; Verlag Von Heinrich Hoff: Mannheim, Germany, 1835; Volume 4, p. 476. [Google Scholar]
  27. Hellström, J.; Sinkkonen, J.; Karonen, M.; Mattila, P. Isolation and structure elucidation of procyanidin oligomers from saskatoon berries (Amelanchier alnifolia). J. Agric. Food Chem. 2007, 55, 157–164. [Google Scholar] [CrossRef] [PubMed]
  28. Karonen, M.; Loponen, J.; Ossipov, V.; Pihlaja, K. Analysis of procyanidins in pine bark with reversed-phase and normal-phase high-performance liquid chromatography–electrospray ionization mass spectrometry. Anal. Chim. Acta 2004, 522, 105–112. [Google Scholar] [CrossRef]
  29. Ado, M.A.; Abas, F.; Ismail, I.S.; Ghazali, H.M.; Shaari, K. Chemical profile and antiacetylcholinesterase, antityrosinase, antioxidant and α-glucosidase inhibitory activity of Cynometra cauliflora L. leaves. J. Sci. Food Agric. 2015, 95, 635–642. [Google Scholar] [CrossRef] [PubMed]
  30. Moura, F.C.S.; dos Santos Machado, C.L.; Paula, F.R.; Couto, A.G.; Ricci, M.; Cechinel-Filho, V.; Bonomini, T.J.; Sandjo, L.P.; Bresolin, T.M.B. Taxifolin stability: In silico prediction and in vitro degradation with HPLC-UV/UPLC–ESI-MS monitoring. J. Pharm. Anal. 2021, 11, 232–240. [Google Scholar] [CrossRef] [PubMed]
  31. Ye, L.H.; He, X.X.; Yan, M.Z.; Chang, Q. Identification of in vivo components in rats after oral administration of lotus leaf flavonoids using ultra fast liquid chromatography with tandem mass spectrometry. Anal. Methods 2014, 6, 6088–6094. [Google Scholar] [CrossRef]
  32. Guimarães, R.; Barros, L.; Dueñas, M.; Carvalho, A.M.; Queiroz, M.J.R.; Santos-Buelga, C.; Ferreira, I.C. Characterisation of phenolic compounds in wild fruits from Northeastern Portugal. Food Chem. 2013, 141, 3721–3730. [Google Scholar] [CrossRef]
  33. Pascale, R.; Acquavia, M.A.; Cataldi, T.R.I.; Onzo, A.; Coviello, D.; Bufo, S.A.; Scrano, L.; Ciriello, R.; Guerrieri, A.; Bianco, G. Profiling of quercetin glycosides and acyl glycosides in sun-dried peperoni di Senise peppers (Capsicum annuum L.) by a combination of LC-ESI(-)-MS/MS and polarity prediction in reversed-phase separations. Anal. Bioanal. Chem. 2020, 412, 3005–3015. [Google Scholar] [CrossRef] [PubMed]
  34. National Center for Biotechnology Information. PubChem Compound Summary for CID 10095180, Kaempferol 7-O-Glucoside. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/Kaempferol-7-o-D-glucopyranoside (accessed on 17 October 2023).
  35. Hamed, A.I.; Al-Ayed, A.S.; Moldoch, J.; Piacente, S.; Oleszek, W.; Stochmal, A. Profiles analysis of proanthocyanidins in the argun nut (Medemia argun—An ancient Egyptian palm) by LC–ESI–MS/MS. J. Mass Spectrom. 2014, 49, 306–315. [Google Scholar] [CrossRef]
  36. Navarro, M.; Moreira, I.; Arnaez, E.; Quesada, S.; Azofeifa, G.; Vargas, F.; Alvarado, D.; Chen, P. Polyphenolic characterization and antioxidant activity of Malus domestica and Prunus domestica cultivars from Costa Rica. Foods 2018, 7, 15. [Google Scholar] [CrossRef]
  37. National Center for Biotechnology Information. PubChem Compound Summary for CID 45360047, Quercetin-3-O-Pentosyl-Pentoside. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/quercetin-3-O-pentosyl-pentoside (accessed on 17 October 2023).
  38. National Center for Biotechnology Information. PubChem Compound Summary for CID 119258, Astilbin. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/Astilbin (accessed on 17 October 2023).
  39. Moharram, F.A.; Marzouk, M.S.A.; Ibrahim, M.T.; Mabry, T.J. Antioxidant galloylated flavonol glycosides from Calliandra haematocephala. Nat. Prod. Res. 2006, 20, 927–934. [Google Scholar] [CrossRef]
  40. Silva, O.; Serrano, R. Micromorphological studies for application in the quality control of herbal medicines-some data of plants used in Mozambique. In Microscopy and Imaging Science: Practical Approaches to Applied Research and Education; Méndez-Vilas, A., Ed.; Formatex Research Center: Badajoz, Spain, 2017; pp. 231–236. [Google Scholar]
  41. Saenger, P.; West, P.W. Determinants of some leaf characteristics of Australian mangroves. Bot. J. Linn. Soc. 2016, 180, 530–541. [Google Scholar] [CrossRef]
  42. Pan, A.D.; Jacobs, B.F.; Herendeen, P.S. Detarieae sensu lato (Fabaceae) from the late Oligocene (27.23 Ma) Guang river flora of north-western Ethiopia: Oligocene ethiopian legumes. Bot. J. Linn. Soc. 2010, 163, 44–54. [Google Scholar] [CrossRef]
  43. Appleton, J. Evaluating the bioavailability of isoquercetin. Nat. Med. J. 2010, 2, 1–6. Available online: https://www.naturalmedicinejournal.com/journal/evaluating-bioavailability-isoquercetin (accessed on 25 January 2023).
  44. Lesjak, M.; Beara, I.; Simin, N.; Pintać, D.; Majkić, T.; Bekvalac, K.; Orčić, D.; Mimica-Dukić, N. Antioxidant and anti-inflammatory activities of quercetin and its derivatives. J. Funct. Foods 2018, 40, 68–75. [Google Scholar] [CrossRef]
  45. Hsieh, H.L.; Yu, M.C.; Cheng, L.C.; Chu, M.Y.; Huang, T.H.; Yeh, T.S.; Tsai, M.M. Quercetin exerts anti-inflammatory effects via inhibiting tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in normal human gastric epithelial cells. World J. Gastroenterol. 2022, 28, 1139. [Google Scholar] [CrossRef] [PubMed]
  46. Aghababaei, F.; Hadidi, M. Recent advances in potential health benefits of quercetin. Pharmaceuticals 2023, 16, 1020. [Google Scholar] [CrossRef] [PubMed]
  47. Nile, A.; Nile, S.H.; Shin, J.; Park, G.; Oh, J.-W. Quercetin-3-Glucoside extracted from apple Pomace induces cell cycle arrest and apoptosis by increasing intracellular ROS levels. Int. J. Mol. Sci. 2021, 22, 10749. [Google Scholar] [CrossRef]
  48. Wang, Y.-H.; Wang, W.-Y.; Chang, C.-C.; Liou, K.-T.; Sung, Y.-J.; Liao, J.-F.; Chen, C.F.; Chang, S.; Hou, Y.C.; Chou, Y.C.; et al. Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation. J. Biomed. Sci. 2006, 13, 127–141. [Google Scholar] [CrossRef] [PubMed]
  49. Lei, L.; Chai, Y.; Lin, H.; Chen, C.; Zhao, M.; Xiong, W.; Zhuang, J.; Fan, X. Dihydroquercetin activates AMPK/Nrf2/HO-1 signaling in macrophages and attenuates inflammation in LPS-induced endotoxemic mice. Front. Pharmacol. 2020, 11, 662. [Google Scholar] [CrossRef] [PubMed]
  50. Ado, M.A.; Mediani, A.; Ismail, I.S.M.; Ghazali, H.M.; Abas, F. Flavonoids from Cynometra cauliflora and their antioxidant, α-glucosidase, and cholinesterase inhibitory activities. Chem. Nat. Compd. 2019, 55, 112–114. [Google Scholar] [CrossRef]
  51. Rauf, A.; Imran, M.; Abu-Izneid, T.; Patel, S.; Pan, X.; Naz, S.; Silva, A.S.; Saeed, F.; Suleria, H.A.R. Proanthocyanidins: A comprehensive review. Biomed. Pharmacother. 2019, 116, 108999. [Google Scholar] [CrossRef] [PubMed]
  52. Kampa, M.; Theodoropoulou, K.; Mavromati, F.; Pelekanou, V.; Notas, G.; Lagoudaki, E.D.; Nifli, A.P.; Morel-Salmi, C.; Stathopoulos, E.N.; Vercauteren, J.; et al. Novel oligomeric proanthocyanidin derivatives interact with membrane androgen sites and induce regression of hormone-independent prostate cancer. J. Pharmacol. Exp. Ther. 2011, 337, 24–32. [Google Scholar] [CrossRef] [PubMed]
  53. Kolodziej, H.; Kayser, O.; Latté, K.P.; Ferreira, D. Evaluation of the antimicrobial potency of tannins and related compounds using the microdilution broth method. Planta Med. 1999, 65, 444–446. [Google Scholar] [CrossRef] [PubMed]
  54. Barbe, A.; Ramé, C.; Mellouk, N.; Estienne, A.; Bongrani, A.; Brossaud, A.; Riva, A.; Guérif, F.; Froment, P.; Dupont, J. Effects of grape seed extract and proanthocyanidin B2 on in vitro proliferation, viability, steroidogenesis, oxidative stress, and cell signaling in human granulosa cells. Int. J. Mol. Sci. 2019, 20, 4215. [Google Scholar] [CrossRef] [PubMed]
  55. Thuan, N.H.; Malla, S.; Trung, N.T.; Dhakal, D.; Pokhrel, A.R.; Chu, L.L.; Sohng, J.K. Microbial production of astilbin, a bioactive rhamnosylated flavanonol, from taxifolin. World J. Microbiol. Biotechnol. 2017, 33, 36. [Google Scholar] [CrossRef] [PubMed]
  56. Paes, J.B.; Diniz, C.E.F.; Marinho, I.V.; de Lima, C.R. Tannin potencial evaluation of six forest species of Brazilian semi-arid region. Cerne 2006, 12, 232–238. [Google Scholar]
  57. Moura, V.M.; Silva, W.C.R.; Raposo, J.D.A.; Freitas-de-Sousa, L.A.; Dos-Santos, M.C.; Oliveira, R.B.; Veras Mourao, R.H. The inhibitory potential of the condensed-tannin-rich fraction of Plathymenia reticulata Benth. (Fabaceae) against Bothrops atrox envenomation. J. Ethnopharmacol. 2016, 183, 136–142. [Google Scholar] [CrossRef]
  58. Oliveira, F.G.D.S.; Veras, B.O.D.; Silva, A.P.S.A.D.; Araújo, A.D.D.; Barbosa, D.C.D.S.; Silva, T.D.C.M.; Ribeiro, E.R.F.R.; Maia, M.M.L.; Júnior, U.P.S.; Lima, V.L.D.M.; et al. Photoprotective activity and HPLC-MS-ESI-IT profile of flavonoids from the barks of Hymenaea martiana Hayne (Fabaceae): Development of topical formulations containing the hydroalcoholic extract. Biotechnol. Biotechnol. Equip. 2021, 35, 504–516. [Google Scholar] [CrossRef]
  59. Landim, L.P.; Feitoza, G.S.; da Costa, J.G. Development and validation of a HPLC method for the quantification of three flavonoids in a crude extract of Dimorphandra gardneriana. Rev. Bras. Farmacogn. 2013, 23, 58–64. [Google Scholar] [CrossRef]
  60. Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L. Polyphenols: Food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727–747. [Google Scholar] [CrossRef]
  61. Tapiero, H.; Tew, K.D.; Ba, G.N.; Mathe, G. Polyphenols: Do they play a role in the prevention of human pathologies? Biomed. Pharmacother. 2002, 56, 200–207. [Google Scholar] [CrossRef] [PubMed]
  62. Chiou, Y.S.; Wu, J.C.; Huang, Q.; Shahidi, F.; Wang, Y.J.; Ho, C.T.; Pan, M.H. Metabolic and colonic microbiota transformation may enhance the bioactivities of dietary polyphenols. J. Funct. Foods 2014, 7, 3–25. [Google Scholar] [CrossRef]
  63. Xiong, H.-H.; Lin, S.-Y.; Chen, L.-L.; Ouyang, K.-H.; Wang, W.-J. The Interaction between Flavonoids and Intestinal Microbes: A Review. Foods 2023, 12, 320. [Google Scholar] [CrossRef] [PubMed]
  64. Graefe, E.U.; Wittig, J.; Mueller, S.; Riethling, A.K.; Uehleke, B.; Drewelow, B.; Pforte, H.; Jacobasch, G.; Derendorf, H.; Veit, M. Pharmacokinetics and bioavailability of quercetin glycosides in humans. J. Clin. Pharmacol. 2001, 41, 492–499. [Google Scholar] [CrossRef] [PubMed]
  65. Maat, L.; Beyerman, H.C. The Imidazole alkaloids. In The Alkaloids: Chemistry and Pharmacology; Brossi, A., Ed.; Academic Press: Cambridge, MA, USA, 1984; pp. 281–333. [Google Scholar] [CrossRef]
  66. Waterman, P.G.; Faulkner, D.F. Imidazole alkaloids from Cynometra hankei. Phytochemistry 1981, 20, 2765–2767. [Google Scholar] [CrossRef]
  67. Abd Aziz, A.F.; Iqbal, M. Antioxidant activity and phytochemical composition of Cynometra cauliflora. J. Exp. Integr. Med. 2013, 3, 337–341. [Google Scholar] [CrossRef]
  68. Afrin, S.; Pervin, R.; Sabrin, F.; Rony, S.R.; Sohrab, M.H.; Islam, M.E.; Islam, K.D.; Billah, M.M. In vitro antioxidant activity, antimicrobial and preliminary cytotoxic activity of Cynometra ramiflora—A mangrove plant. J. Microbiol. Biotechnol. Food Sci. 2016, 6, 844–850. [Google Scholar] [CrossRef]
  69. Sajid, Z.I.; Anwar, F.; Shabir, G.; Rasul, G.; Alkharfy, K.M.; Gilani, A.H. Antioxidant, antimicrobial properties and phenolics of different solvent extracts from bark, leaves and seeds of Pongamia pinnata (L.) Pierre. Molecules 2012, 17, 3917–3932. [Google Scholar] [CrossRef] [PubMed]
  70. Mulyani, Y.; Syaputra, N.D.; Dewi, K.C.; Lili, W.; Agung, M.U.K. Total phenolic, flavonoid content and antioxidant capacity of stem bark, root, and leaves methanolic extract of Rhizophora mucronata Lam. World News Nat. Sci. 2019, 26, 118–127. [Google Scholar]
  71. Wong, P.L.; Fauzi, N.A.; Mohamed Sumarlin, S.N.; Abdul Hamid, N.A.; Abd Ghafar, S.Z.; Azizan, A.; Zolkeflee, N.K.Z.; Abas, F. Biological activities of selected plants and detection of bioactive compounds from Ardisia elliptica using UHPLC-Q-executive orbitrap mass spectrometry. Molecules 2020, 25, 3067. [Google Scholar] [CrossRef]
  72. Mallawaarachchi, M.A.L.N.; Madhujith, T.; Suriyagoda, L.D.B.; Pushpakumara, D.K.N.G. Antioxidant efficacy of selected underutilized fruit species grown in Sri Lanka. Trop. Agric. Res. 2021, 32, 68–80. [Google Scholar] [CrossRef]
  73. European Directorate for the Quality of Medicines, EDQM. European Pharmacopoeia, 10th ed.; Directorate for the Quality of Medicines, Council of Europe: Strasbourg, France, 2019. [Google Scholar]
  74. Nipun, T.S.; Khatib, A.; Ahmed, Q.U.; Nasir, M.H.M.; Supandi, F.; Taher, M.; Saiman, M.Z. Preliminary Phytochemical screening, in vitro antidiabetic, antioxidant activities, and toxicity of leaf extracts of Psychotria malayana Jack. Plants 2021, 10, 2688. [Google Scholar] [CrossRef] [PubMed]
  75. Le Thi, V.A.; Nguyen, N.L.; Nguyen, Q.H.; Van Dong, Q.; Do, T.Y.; Nguyen, T.K.O. Phytochemical screening and potential antibacterial activity of defatted and nondefatted methanolic extracts of Xao Tam Phan (Paramignya trimera (Oliv.) Guillaum) peels against multidrug-resistant bacteria. Scientifica 2021, 2021, 4233615. [Google Scholar] [CrossRef] [PubMed]
  76. Dhivya, R.; Manimegalai, K. Preliminary phytochemical screening and GC-MS profiling of ethanolic flower extract of Calotropis gigantea Linn. (Apocyanaceae). J Pharmacogn. Phytochem. 2013, 2, 28–32. [Google Scholar]
  77. Trease, G.E.; Evans, W.C. Pharmacognosy, 15th ed.; Saunders Publishers: London, UK, 2002; pp. 214–393. [Google Scholar]
  78. Scalbert, A.; Monties, B.; Janin, G. Tannins in wood: Comparison of different estimation methods. J. Agric. Food Chem. 1989, 37, 1324–1329. [Google Scholar] [CrossRef]
  79. Olivera, F.D.; Vina, S.Z.; Marani, C.M.; Ferreyra, R.M.; Mugridge, A.; Chaves, A.R. Effect of blanching on the quality of Brussels sprouts (Brassica olearacea L. gemmifera DC) after frozen storage. J. Food Eng. 2008, 84, 148–155. [Google Scholar] [CrossRef]
  80. Porter, L.J.; Hrstich, L.N.; Chan, B.G. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 1986, 25, 223–230. [Google Scholar] [CrossRef]
  81. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci Tech. 1995, 28, 25–30. [Google Scholar] [CrossRef]
  82. Benzie, I.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76. [Google Scholar] [CrossRef]
  83. Ramful, D.; Bahorun, T.; Bourdon, E.; Tarnus, E.; Aruoma, O.I. Bioactive phenolics and antioxidant propensity of flavedo extracts of Mauritian citrus fruits: Potential prophylactic ingredients for functional foods application. Toxicology 2010, 278, 75–87. [Google Scholar] [CrossRef]
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Figure 1. Cynometra iripa (a) general aspect; (b) fresh green leaf; (c) fresh bark.
Figure 1. Cynometra iripa (a) general aspect; (b) fresh green leaf; (c) fresh bark.
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Figure 2. C. iripa leaf macroscopic characters. (a) dried leaflet; (b) abaxial and adaxial view of the asymmetrical leaf. Scale bars: (a,b) = 1 cm.
Figure 2. C. iripa leaf macroscopic characters. (a) dried leaflet; (b) abaxial and adaxial view of the asymmetrical leaf. Scale bars: (a,b) = 1 cm.
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Figure 3. C. iripa leaf macroscopic characters. Details of (a) adaxial and (b) abaxial view of emarginated apex; (c) cuneate base, petiolules, and rachis; (d) hairy petiolule. Scale bars: (a–c) = 5 mm; (d) = 2 mm.
Figure 3. C. iripa leaf macroscopic characters. Details of (a) adaxial and (b) abaxial view of emarginated apex; (c) cuneate base, petiolules, and rachis; (d) hairy petiolule. Scale bars: (a–c) = 5 mm; (d) = 2 mm.
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Figure 4. C. iripa dried leaf. (a,b) LM micrographs of transverse section. Details of central vein and mesophyll; (c) adaxial surface without stomata; (d) abaxial surface with paracytic stomata; (e) calcium oxalate prismatic crystals at the vein; (f) unicellular trichomes on petiolule; xy—xylem, ph—phloem, sc—sclerenchyma, ade—adaxial epidermis, pp—palisade parenchyma, abe—abaxial epidermis. Scale bars: (a) = 100 µm; (b–f) = 50 µm.
Figure 4. C. iripa dried leaf. (a,b) LM micrographs of transverse section. Details of central vein and mesophyll; (c) adaxial surface without stomata; (d) abaxial surface with paracytic stomata; (e) calcium oxalate prismatic crystals at the vein; (f) unicellular trichomes on petiolule; xy—xylem, ph—phloem, sc—sclerenchyma, ade—adaxial epidermis, pp—palisade parenchyma, abe—abaxial epidermis. Scale bars: (a) = 100 µm; (b–f) = 50 µm.
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Figure 5. C. iripa powdered leaf. Details of (a) fragments of epidermal cells and palisade parenchyma; (b) fibers; (c) free prismatic calcium oxalate crystals; (d) unicellular non-glandular trichome. Scale bars: (a–d) = 50 µm.
Figure 5. C. iripa powdered leaf. Details of (a) fragments of epidermal cells and palisade parenchyma; (b) fibers; (c) free prismatic calcium oxalate crystals; (d) unicellular non-glandular trichome. Scale bars: (a–d) = 50 µm.
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Figure 6. C. iripa bark macroscopic characters. Details of (a) adaxial view; (b) abaxial view. Scale bars: (a,b) = 1 cm.
Figure 6. C. iripa bark macroscopic characters. Details of (a) adaxial view; (b) abaxial view. Scale bars: (a,b) = 1 cm.
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Figure 7. Microscopic characters of C. iripa bark. Details of (a–d) transverse section of bark showing lenticel (le), periderm (pe), phelloderm (phe), and cortex; layer of thick sclereids (scl) and parenchyma cell (pa); a group of sclereids (scl); medullary rays (med) with 2–5 cell layers in secondary phloem; longitudinal sections (e,f) showing fibers (fb), calcium oxalate prismatic crystals (pc), starch granules (stg), at parenchyma (e); calcium oxalate prismatic crystals (pc) associated with fibers (f). Scale bars: (a) = 200 µm; (b,d,e) = 100 µm; (c,f) = 50 µm.
Figure 7. Microscopic characters of C. iripa bark. Details of (a–d) transverse section of bark showing lenticel (le), periderm (pe), phelloderm (phe), and cortex; layer of thick sclereids (scl) and parenchyma cell (pa); a group of sclereids (scl); medullary rays (med) with 2–5 cell layers in secondary phloem; longitudinal sections (e,f) showing fibers (fb), calcium oxalate prismatic crystals (pc), starch granules (stg), at parenchyma (e); calcium oxalate prismatic crystals (pc) associated with fibers (f). Scale bars: (a) = 200 µm; (b,d,e) = 100 µm; (c,f) = 50 µm.
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Figure 8. C. iripa powdered bark. Details of (a) fragments of fibers; (b) fragments of parenchyma (pf) and periderm (pef); (c) scattered calcium oxalate prismatic crystals (pc); scale bars: (a–c) = 50 µm.
Figure 8. C. iripa powdered bark. Details of (a) fragments of fibers; (b) fragments of parenchyma (pf) and periderm (pef); (c) scattered calcium oxalate prismatic crystals (pc); scale bars: (a–c) = 50 µm.
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Figure 9. LC/MS chromatographic profile of main marker compounds in C. iripa leaf (CIL) and C. iripa bark (CIB).
Figure 9. LC/MS chromatographic profile of main marker compounds in C. iripa leaf (CIL) and C. iripa bark (CIB).
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Figure 10. Major identified compounds from leaf (a,b) and bark (c,d) hydroethanolic extracts.
Figure 10. Major identified compounds from leaf (a,b) and bark (c,d) hydroethanolic extracts.
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Figure 11. Scavenging activity of C. iripa leaf (CIL) and C. iripa bark (CIB) extracts; ASC-ascorbic acid.
Figure 11. Scavenging activity of C. iripa leaf (CIL) and C. iripa bark (CIB) extracts; ASC-ascorbic acid.
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Figure 12. The ferric reducing capacity of C. iripa leaf (CIL) and C. iripa bark (CIB) extracts; ASC-ascorbic acid.
Figure 12. The ferric reducing capacity of C. iripa leaf (CIL) and C. iripa bark (CIB) extracts; ASC-ascorbic acid.
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Figure 13. Map of Bangladesh showing plant collection area in the Sundarbans mangrove forest; ca.1—collection area 1: Koromjol and ca.2—collection area 2: Harbaria eco-tourism center, Bangladesh.
Figure 13. Map of Bangladesh showing plant collection area in the Sundarbans mangrove forest; ca.1—collection area 1: Koromjol and ca.2—collection area 2: Harbaria eco-tourism center, Bangladesh.
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Table 1. Previously reported chemical constituents identified in Cynometra iripa.
Table 1. Previously reported chemical constituents identified in Cynometra iripa.
Plant PartChemical ClassCompounds NameRef.
Leaf, seed oilFatty acidsleaf: arachidic acid, behenic acid, cis-11-eicosenoic acid, lauric acid, linolenic acid, myristic acid, oleic acid, pentadecanoic acid, palmitic acid, stearic acid[18]
seed: arachidic acid, behenic acid, caproic acid, cis-8, 11, 14-eicosatrienoic acid, cis-13, 16-docosadienoic acid, lauric acid, lignoceric acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, tricosanoic acid.
Seed, seed coatTerpenoidscholesta-4,6-dien-3-beta-ol, β-carotene, β-sitosterol, stigmast-4-en-3-one, squalene [19,20]
Esters1,2-benzene dicarboxylic acid mono (2-ethylhexyl) ester, butyric acid-2-pentadecyl ester, 1,2-benzene dicarboxylic acid butyl-2-ethylhexyl ester
Fatty alcohols1-eicosanol, falcarinol
Phenols2,5-di-tert-butyl-1,4-benzoquinone, 3,5-di-tert-butyl-4-hydroxybenzaldehyde
Vitaminsvitamin E
Table 2. Quantitative microscopy characteristics of C. iripa leaf and C. iripa bark.
Table 2. Quantitative microscopy characteristics of C. iripa leaf and C. iripa bark.
Anatomical CharacteristicsMin–MaxMean ± SDMedian
Leaf
Total stomata dimensions (abaxial surface)
   Length (µm)16.01–28.8520.55 ± 2.8719.66
   Width (µm)14.62–21.2916.90 ± 1.8316.36
Adaxial epidermal cell
   Length (µm)10.26–18.6413.53 ± 2.5013.24
   Width (µm)5.45–13.418.52 ± 1.798.52
Abaxial epidermal cell
   Length (µm)12.84–34.2020.84 ± 5.9718.86
   Width (µm)8.37–15.9511.33 ± 1.7011.18
Cross-sectional Features
Total mesophyll length (µm)108.38–164.37132.25 ± 15.24131.62
Xylem vessel diameter (µm)9.19–21.2113.50 ± 3.4312.31
Calcium oxalate prismatic crystals width (µm)3.53–8.705.89 ± 1.326.19
Stomatal Index20.15 ± 3.44
Bark
Schlerid cell wall thickness (µm)4.62–11.507.65 ± 2.157.13
Starch diameter (µm)3.93–7.956.24 ± 1.056.37
Calcium oxalate prismatic crystals width (µm)4.90–13.128.24 ± 1.577.86
Abbreviations: Min—minimum; Max—maximum; SD—standard deviation.
Table 3. C. iripa yield of extraction and drug extract ratio.
Table 3. C. iripa yield of extraction and drug extract ratio.
Parts UsedWeight (g)Yield of Extract in %DER (m/m)
CIL20014.96.71:1
CIB17.65.68:1
Abbreviations: CIL—C. iripa leaf; CIB—C. iripa bark; DER—drug extract ratio.
Table 4. Preliminary phytochemical screening on C. iripa leaf and bark extracts.
Table 4. Preliminary phytochemical screening on C. iripa leaf and bark extracts.
PhytoconstituentsTestResults
CILCIB
AlkaloidsBouchardat/Mayer/Dragendorff−−
Phenolic compoundsFerric chloride test++
Acetic acid test++
SaponinsFoam test++
Abbreviations: CIL—C. iripa leaf; CIB—C. iripa bark; + (positive); − (negative).
Table 5. LC-UV/DAD-ESI/MS identification of the main marker compound of C. iripa leaf extracts.
Table 5. LC-UV/DAD-ESI/MS identification of the main marker compound of C. iripa leaf extracts.
Plant PartPeaktR
(min)		λmax (nm)MW[M − H]− (m/z)Fragment Ions
(m/z)		Tentative Assignment
CILa27.77218, 223, 27911551154865, 577, 425, 287B-type proanthocyanidin (tetramer)
b30.39218, 223, 279866865577, 289B-type proanthocyanidin (trimer)
c31.08218, 223, 279144314421154, 865, 577, 289B-type proanthocyanidin (pentamer 1)
d31.95218, 223, 279144314421154, 865, 577, 289B-type proanthocyanidin (pentamer 2)
e34.20223, 284436435303, 285, 151taxifolin pentoside isomer 1
f36.48223, 285436435303, 285, 151taxifolin pentoside isomer 2
g37.76223, 268, 284, 351464463435, 303, 285, 152quercetin 3-O-glucoside
h38.67223, 289436435303, 285, 151taxifolin pentoside isomer 3
i40.53218, 268, 284, 351434433301quercetin 7-O-pentoside/apioside
j41.27218, 268, 284, 347448447285kaempferol 7-O-glucoside
k58.85223, 269, 334270269269, 89apigenin
Abbreviations: CIL—C. iripa leaf; m/z—mass to charge ratio [M − H]−—negative mass electrospray ionization mode; tR—retention time; λmax—wavelength of maximum absorbance.
Table 6. LC-UV/DAD-ESI/MS identification of the main marker compound of C. iripa bark extracts.
Table 6. LC-UV/DAD-ESI/MS identification of the main marker compound of C. iripa bark extracts.
Plant PartPeaktR
(min)		λmax (nm)MW[M − H]− (m/z)Fragment Ions
(m/z)		Tentative Assignment
CIBa′27.76221, 27911551154865, 577, 425, 289B-type proanthocyanidin (tetramer 1)
b′30.38221, 280866865577, 289B-type proanthocyanidin (trimer 1)
c′31.08221, 28011551154865, 577, 425, 289B-type proanthocyanidin (tetramer 2)
d′34.78221, 227, 280, 320562561433, 287, 273B-type proanthocyanidin (dimer 1)
e′35.32221, 235, 280, 320562561433, 287, 271B-type proanthocyanidin (dimer 2)
f′36.47221, 281, 346566565301quercetin-3-O-pentosyl-pentoside
g′37.87221, 227, 286450449303, 285, 151taxifolin 3-O-rhamnoside 1
h′38.60221, 227, 287450449303, 285, 151taxifolin 3-O-rhamnoside 2
i′40.18221, 227, 284450449303, 285, 151taxifolin 3-O-rhamnoside 3
j′41.33221, 228, 280, 350600599447, 301quercitrin 3″-O-gallate
k′58.85223, 269, 334270269269, 89apigenin
Abbreviations: CIB—C. iripa bark; m/z—mass to charge ratio [M − H]−—negative mass electrospray ionization mode; tR—retention time; λmax—wavelength of maximum absorbance.
Table 7. Quantification of the main class of secondary metabolites of C. iripa leaf and bark extracts.
Table 7. Quantification of the main class of secondary metabolites of C. iripa leaf and bark extracts.
Plant ExtractsTPC (mg GAE/g)TFC (mg CE/g)TCTC (mg CCE/g)
CIL1521 ± 4.7164 ± 1.00755 ± 4.00
CIB1476 ± 4.0982 ± 0.581021 ± 5.51
Abbreviations: TPC—total phenolic content; TFC—total flavonoid content; TCTC—total condensed tannin content; GAE—gallic acid equivalents; CE—catechin equivalents; CCE—cyanidine chloride equivalents; SD—standard deviation. Each value in the table is represented as mean ± SD.
Table 8. Correlation between C. iripa leaf and C. iripa bark phenolic content and antioxidant activity.
Table 8. Correlation between C. iripa leaf and C. iripa bark phenolic content and antioxidant activity.
CIL
VariablesTPCTFCTCTCDPPHFRAP
TPC1
TFC0.961
TCTC0.971.001
DPPH0.850.960.941
FRAP0.920.990.990.991
CIB
VariablesTPCTFCTCTCDPPHFRAP
TPC1
TFC0.991
TCTC0.9911
DPPH1.000.990.991
FRAP0.950.990.990.961
Abbreviations: CIL—C. iripa leaf; CIB—C. iripa bark; TPC—total phenolic content; TFC—total flavonoid content; TCTC—total condensed tannin content; DPPH—2,2-Diphenyl-1-picrylhydrazyl; FRAP—ferric-reducing antioxidant power.
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Sabiha, S.; Hasan, K.; Lima, K.; Malmir, M.; Serrano, R.; Moreira da Silva, I.; Rocha, J.; Islam, N.; Silva, O. Quality Studies on Cynometra iripa Leaf and Bark as Herbal Medicines. Molecules 2024, 29, 2629. https://doi.org/10.3390/molecules29112629

AMA Style

Sabiha S, Hasan K, Lima K, Malmir M, Serrano R, Moreira da Silva I, Rocha J, Islam N, Silva O. Quality Studies on Cynometra iripa Leaf and Bark as Herbal Medicines. Molecules. 2024; 29(11):2629. https://doi.org/10.3390/molecules29112629

Chicago/Turabian Style

Sabiha, Shabnam, Kamrul Hasan, Katelene Lima, Maryam Malmir, Rita Serrano, Isabel Moreira da Silva, João Rocha, Nurul Islam, and Olga Silva. 2024. "Quality Studies on Cynometra iripa Leaf and Bark as Herbal Medicines" Molecules 29, no. 11: 2629. https://doi.org/10.3390/molecules29112629

APA Style

Sabiha, S., Hasan, K., Lima, K., Malmir, M., Serrano, R., Moreira da Silva, I., Rocha, J., Islam, N., & Silva, O. (2024). Quality Studies on Cynometra iripa Leaf and Bark as Herbal Medicines. Molecules, 29(11), 2629. https://doi.org/10.3390/molecules29112629

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