Quality Studies on Cynometra iripa Leaf and Bark as Herbal Medicines

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.


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 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].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].Leaf, seed oil Fatty acids leaf: 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.
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.

Leaf Macroscopic Characteristics
The macroscopic observation (Figures 2 and 3) revealed a dried grey color asymmetrical leaflet, with leaf lamina 3.0-6.6cm in length and 1.4-2.8cm 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).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 nonglandular 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).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 nonglandular 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).

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).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).

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.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 (1.65-5.96µm), 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.

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,

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.Abbreviations: Min-minimum; Max-maximum; SD-standard deviation.

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.

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.

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 Tables 5 and 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.

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 Tables 5 and 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.Based on the differences between monomer units, UV spectra, and fragmentation pattern, these compounds were also identified as B-type proanthocyanidin pentamers. .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).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.

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.

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.

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.

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.

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 Fe 2+ /g dry weight, and of the CIB extract, this is 77.94 ± 2.02 µmol Fe 2+ /g dry weight, while the FRAP value of the quercetin and ASC was 121.51 ± 0.94 µmol Fe 2+ /g dry weight and 149.84 ± 1.08 µmol Fe 2+ /g dry weight, respectively.Comparatively, the CIB extract was shown to have more antioxidant potential than CIL.

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 In fact, the IC 50 (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.

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 Fe 2+ /g dry weight, and of the CIB extract, this is 77.94 ± 2.02 µmol Fe 2+ /g dry weight, while the FRAP value of the quercetin and ASC was 121.51 ± 0.94 µmol Fe 2+ /g dry weight and 149.84 ± 1.08 µmol Fe 2+ /g dry weight, respectively.Comparatively, the CIB extract was shown to have more antioxidant potential than CIL.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.

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 Fe 2+ /g dry weight, and of the CIB extract, this is 77.94 ± 2.02 µmol Fe 2+ /g dry weight, while the FRAP value of the quercetin and ASC was 121.51 ± 0.94 µmol Fe 2+ /g dry weight and 149.84 ± 1.08 µmol Fe 2+ /g dry weight, respectively.Comparatively, the CIB extract was shown to have more antioxidant potential than CIL.

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

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 made an outstanding contribution to the antioxidant activity of the CIL and CIB extracts.

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.
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.
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].
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 IC 50 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 Fe 2+ /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 IC 50 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 IC 50 value of 0.47 ± 0.03 g of dry weight/mL and a FRAP assay with reducing power of 25.07 ± 0.73 µmol Fe 2+ /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.

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.
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 Fe 2+ /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.

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.

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].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.

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].

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).

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].

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) = Wt 1 /Wt 2 × 100% Wt 1 and Wt 2 represent the final weight of the dried extract and the primary weight of the leaf/bark powder [74].

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.

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 Na 2 CO 3 (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% NaNO 2 were added, and the mixture was left to incubate for 5 min.After that, 150 µL of 10% AlCl 3 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.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 = A 0 − A 1 /A 0 × 100 where A 0 = the absorbance of the control and A 1 = the absorbance of the standard.The IC 50 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.
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.

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).

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.

Figure 3 .
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.Microscopic Characteristics Light microscopy (LM) analysis of transversal sections of C. iripa leaf showed rectangular to polygonal upper epidermis cells, smaller (10.26-18.64µm) 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 (Figure4c,d).Calcium oxalate prismatic crystals (3.52-8.70 µm) were observed in the veins (Figure4e), and unicellular and pointed nonglandular trichomes were observed in the petiolules (Figure4f).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 (Figure5a-d).

Figure 9 .
Figure 9. LC/MS chromatographic profile of main marker compounds in C. iripa leaf (CIL) and C. iripa bark (CIB).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

Figure 9 .
Figure 9. LC/MS chromatographic profile of main marker compounds in C. iripa leaf (CIL) and C. iripa bark (CIB).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.
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 (C 5 H 10 O 5 ) 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) 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.

Table 2 .
Quantitative microscopy characteristics of C. iripa leaf and C. iripa bark.

Table 3 .
C. iripa yield of extraction and drug extract ratio.

Table 4 .
Preliminary phytochemical screening on C. iripa leaf and bark extracts.

Table 5 .
LC-UV/DAD-ESI/MS identification of the main marker compound of C. iripa leaf extracts.: CIL-C.iripa leaf; m/z-mass to charge ratio [M − H] − -negative mass electrospray ionization mode; t R -retention time; λ max -wavelength of maximum absorbance. Abbreviations

Table 6
presents data on the main compounds identified from C.

Table 6 .
LC-UV/DAD-ESI/MS identification of the main marker compound of C. iripa bark extracts.

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.

Table 8 .
Correlation between C. iripa leaf and C. iripa bark phenolic content and antioxidant activity.