**Isolation, Structure Elucidation and Biological Activity of Natural Products**

Editor

**Jacqueline Aparecida Takahashi**

Basel • Beijing • Wuhan • Barcelona • Belgrade • Novi Sad • Cluj • Manchester

*Editor* Jacqueline Aparecida Takahashi Department of Chemistry Universidade Federal de Minas Gerais Belo Horizonte Brazil

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Molecules* (ISSN 1420-3049) (available at: www.mdpi.com/journal/molecules/special issues/Np isolation).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

Lastname, A.A.; Lastname, B.B. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-8973-2 (Hbk) ISBN 978-3-0365-8972-5 (PDF) doi.org/10.3390/books978-3-0365-8972-5**

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) license.

## **Contents**


Reprinted from: *Molecules* **2022**, *27*, 1506, doi:10.3390/molecules27051506 . . . . . . . . . . . . . . **103**


## **About the Editor**

## **Jacqueline Aparecida Takahashi**

Jacqueline Aparecida Takahashi has held the position of Professor of Organic Chemistry since 1996 at the Universidade Federal de Minas Gerais (UFMG, Brazil). UFMG is one of the major public universities mantained by the Brazilian Government and has over 50,000 students distributed in 91 undegraduate courses and 90 Graduate programs. Prof. Takahashi has a Bachelor's Degree in Industrial Pharmacy, and a PhD in Science/Organic Chemistry, both from UFMG, and worked for two years at the University of Sussex (Brighton, UK) under the supervision of Prof. James Hanson during her PhD. She spent one year as a Research Scholar at the University of Arizona (Tucson, US) in the group of Prof. Leslie Gunatilaka. Her main research interests over the last 30 years have mainly been the use of fungi to produce biologically active natural products and the microbial functionalization of terpenes (biotransformations), as well as plants of the Brazilian biodiversity. Lately she has also dedicated time to study Brazilian and International Laws on the use and preservation of Brazilian and world biodiversity, including aspects related to climate changes, traditional knowledge and regulatory issues.

## **Preface**

The Special Issue "Isolation, Structure Elucidation and Biological Activity of Natural Products" was proposed to bring together work and reviews on natural products of plant and microbial origin, with special interest in the biological activity of these products. With the various current problems related to the loss of biodiversity associated with climate change and the exploitation of natural areas for economic activities, it is urgent to show the role biodiversity plays for the scientific community and society. Based on this context, the contributions published in this Issue show several works refarding the possible industrial applications of plants and microorganisms, while the review articles show the vastness of existing natural products, many of which deserve further studies for technological development. This Issue is addressed to natural product chemistry researchers, including young researchers enthusiastic about the potential of biodiversity for diverse applications. Likewise, authorities interested in innovation in the area of natural products, companies, and industries will be able to benefit from the articles published in this Issue. I would like to thank the authors of the published articles, as well as the editorial assistance and MDPI's invitation for me to act as guest editor of this Issue.

> **Jacqueline Aparecida Takahashi** *Editor*

## *Editorial* **Special Issue—"Isolation, Structure Elucidation and Biological Activity of Natural Products"**

**Jacqueline Aparecida Takahashi**

Exact Sciences Institute, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, 6627, Belo Horizonte 31270-901, Brazil; takahashi.ufmg@gmail.com

This Special Issue of *Molecules* gathers fourteen research studies and three review papers covering developments in the scope of the isolation, structure elucidation and biological activity of natural products. Plants are undoubtedly the most well-known sources of bioactive natural products, since plants have been used medicinally by traditional communities for centuries, defining their specific benefits to human health. In this Special Issue, the plants addressed are *Bauhinia forficata*, *Placolobium vietnamense*, *Tamarix chinensis*, *Peperomia obtusifolia*, *Cymbidium ensifolium*, *Dendrobium delacourii*, *Turnera subulata*, *Eruca sativa*, *Miconia chamissois* and *Persea americana*, with the aim of achieving the identification of their chemical compositions, the prospection of biological activities, the standardization of extracts and the use of agro-industrial residues. Some studies involved hyphenated techniques such as UPLC-IT-MS<sup>n</sup> [1], UHPLC-ESI-QTOF [2], UHPLC/UV/MS/MS [3], PSMS [4] and UPLC–MS/MS [5], which are very useful tools in research on natural products.

*B. forficata* is a tropical species popularly used in Brazil to treat type II diabetes, rheumatism, local pain, uric acid and uterine problems. Jung et al (2022) carried out a comparative study between locally collected leaves of this species and commercial samples [6]. Using LC-HRMS, a very useful hyphenated analytical technique, the authors were able to identify the presence of flavonoids, phenolic acids and other phenolic volatile compounds, including flavonoid O-glycosides in the plant, and these metabolites were related to the pharmacological actions reported for *B. forficata.* The plant inhibited the α-amylase enzyme, and the results converged to reinforce the biological and pharmacological potential of *B. forficata* as a hypoglycemiant agent [6]. Studies of this type are of great value, especially considering that *B. forficata* is an easily accessible plant for the population and is already commercialized.

*P. vietnamense,* known in Vietnam as "Rang Rang", is also distributed throughout the world's tropical regions and is used as a folk medicine to treat snakebites, debility and to increase strength after childbirth. Stems of *P. vietnamense* were studied by Do, Huynh, and Sichaem (2022), who successfully isolated eight natural constituents of this species, including a new isoflavone derivative and three new benzyl derivatives, together with four known compounds of the pyranoisoflavone type [7]. The authors conducted a cytotoxic evaluation of the effects of the aforementioned natural products on a human hepatocellular carcinoma (Hep G2) cell line, determined the cytotoxic effects and measured the production of nitric oxide (NO) by RAW 264.7 cells. Among the compounds tested, placovinone A exhibited the most significant cytotoxicity toward the Hep G2 cell line and also strongly inhibited LPS-induced NO production [7]. The authors concluded that placovinone A is a promising lead for discovering potential anticancer and anti-inflammatory agents.

The work of Jiao et al. (2022), conducted with the Chinese plant *T. chinensis*, which is used to treat rheumatoid arthritis, measles and measles complicated with pneumonia, is an excellent example of the success of activity-guided extraction, isolation and purification targeting a specific class of compounds [1]. In this case, the authors were focused on the bioactive polysaccharides present in *T*. *chinensis*. Flavonoids, triterpenoids, organic acids and volatile oils with anti-inflammatory, bacteriostatic, antioxidant and hepatoprotective

**Citation:** Takahashi, J.A. Special Issue—"Isolation, Structure Elucidation and Biological Activity of Natural Products". *Molecules* **2023**, *28*, 5392. https://doi.org/10.3390/ molecules28145392

Received: 10 July 2023 Accepted: 12 July 2023 Published: 14 July 2023

**Copyright:** © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

effects have already been described from this plant. An activity-guided approach, followed by careful spectroscopic study (HPGPC-ELSD, UPLC-IT-MS<sup>n</sup> and NMR analysis), led to the isolation and identification of two novel natural flavonoid-substituted polysaccharides. Both polysaccharides were substituted by quercetin and exhibited anticomplement activities in vitro. Since the authors were focusing on inflammatory responses in viral pneumonia, antioxidant activities were also determined for the polysaccharides. The authors indicated that molecules containing both flavonoids and polysaccharides have advantageous drug delivery. Structure–activity studies showed that multiple monosaccharides also contribute to the anticomplement activity of *T. chinensis* flavonoid-substituted polysaccharides [1].

*P. obtusifolia* is an ornamental plant species that is widely distributed in tropical areas. Plants from the *Peperomia* genus have been traditionally used to treat asthma, gastric ulcers, bacterial infection, pain and inflammation. Ware et al. (2022) isolated two new phenolic compounds from *P. obtusifolia* (peperomic ester and peperoside), along with other five known metabolites that were screened for their anthelmintic (*Caenorhabditis elegans*), antifungal (*Botrytis cinerea*, *Septoria tritici* and *Phytophthora infestans*) and antibacterial (*Bacillus subtilis*, *Aliivibrio fisheri*) activities, as well as for their cytotoxicity against human prostate (PC-3) and colorectal (HT-29) cancer cell lines [8]. One of the metabolites tested, peperobtusin A, strongly affected the viability and growth of PC-3 cells in MTT and a CV fast-screening assay and was moderately active against the HT-29 cell line [8]. The results are an indicative that ornamental plants can also be sources of bioactive metabolites.

The roots of *Cymbidium ensifolium*, another ornamental plant known as "nang kham" or "chulan" in Thailand, are used in traditional Thai medicine to alleviate liver dysfunction and nephropathy. The aerial parts of *C. ensifolium* were studied by Jimoh et al (2022), who isolated three novel dihydrophenanthrene derivatives (cymensifins A, B and C), along with two known compounds, cypripedin and gigantol [9]. The chemical structures of the dihydrophenanthrene derivatives were elucidated, mainly via HMBC and NOESY correlations. Their activity against human lung cancer H460, breast cancer MCF7 and colon cancer CaCo<sup>2</sup> cells were evaluated in cell viability assays and for apoptosis/necrosis. The most promising anticancer compound was cymensifin A, which was found to be active against different cancer cells with higher safety profiles compared with the control, cisplatin [9].

Another orchid, *Dendrobium delacourii*, named "Ueang Dok Ma Kham" in Thailand, was studied by Thant et al. (2022) [10]. They isolated 11 compounds and identified them as hircinol, ephemeranthoquinone, densifloral B, moscatin, 4,9-dimethoxy-2,5-phenanthrenediol, gigantol, batatasin III, lusianthridin, 4,40 ,7,70 -tetrahydroxy-2,20 -dimethoxy-9,90 ,10,100 -tetrahydro-1,1 0 -biphenanthrene, phoyunnanin E and phoyunnanin C. Dimeric phenanthrene derivatives presented stronger α-glucosidase inhibitory activity than the monomers. A kinetic study indicated the active metabolites to be non-competitive enzyme inhibitors. The authors argued the benefits of non-competitive inhibitors over competitive inhibitors. Regarding anti-adipogenic action, ephemeranthoquinone and densifloral B showed the highest levels of activity; the latter restrained adipocyte differentiation in 3T3-L1 cells in a dose-dependent manner [10]. The authors suggested that densifloral B might inhibit adipocyte differentiation via the suppression of the Akt-mediating GSK3β and AMPK–ACC signals. These results are of great interest since diabetes and obesity are major problems worldwide.

Extracts from flowers of the tropical plant *Turnera subulata* were studied by Luz et al. (2022) [5]. Their in vitro immunomodulatory effects in RAW 264.7 macrophages stimulated via LPS were evaluated in the search for anti-inflammatory drugs with low side effects. Vitexin-2-*O*-rhamnoside was identified in the extracts via UPLC–MS/MS, as were methoxyisoflavones, pheophorbides, octadecatrienoic, stearidonic, ferulic acids and some amino acids. The results demonstrate the immunomodulatory effects of aqueous and hydroalcoholic extracts of *T. subulata* flowers and leaves through the inhibition of inflammatory TNF-α and IL-1β cytokine secretion [5]. Increases in anti-inflammatory IL-10 cytokine levels also supported the activity and corroborate the ethnopharmacological use of plants from the *Turnera* genus in folk medicine as anti-inflammatory remedies.

In addition to essentially medicinal and ornamental plants, one of the works published in this Special Issue focused on a plant used mainly as food, *Eruca sativa* (rocket). Based on works using animal models that associated rocket with biological activities, such as antihypertensive, nephroprotective and antidiabetic activities, Teixeira et al. (2022) hypothesized that this plant could have anti-hyperuricemic effects, combatting a causal factor of hypertension and diabetes [2]. Nine compounds were detected via UHPLC-ESI-QTOF: kaempferol-3-O-β-glucoside, kaempferol-3,40 -di-O-β-glucoside, kaempferol-3-O-(2 sinapoyl-β-glucoside)-40 -O-glucoside, glucosativin glucosinolate, glucoraphanin glucosinolate, leucine, tryptophan, angustione and erucamide. Kaempferol-3,40 -di-O-β-glucoside was elegantly characterized via NMR and quantified in the extract. the anti-hyperuricemic activities of the extracts were mainly related to uricosuric action [2]. Although other mechanisms are yet to be elucidated, the authors suggested that the results indicate the potential use of *E. sativa* in the treatment of hyperuricemia and its comorbidities.

Ferreira et al. (2022) raised an important issue in the study of medicinal plants, which is the standardization of extracts and the variations relating to seasonal variations. The authors focused on *Miconia chamissois*, known as "Folha de Bolo", "Sabiazeira", or "Pixirica" in Brazil, a plant associated with antimicrobial and antioxidant activities, the in vitro inhibition of the enzymes tyrosinase and alpha-amylase, the inhibition of MMP-2 and MMP-9 and cytotoxicity against human cervical cancer cell lines [3]. A standardized extract was formulated, and some major constituents, such as apigenin C-glycosides (vitexin/isovitexin), luteolin C-glycosides (orientin/isoorientin), miconioside B, matteucinol-7-O-β-apiofuranosyl (1 → 6)-β-glucopyranoside and farrerol, were identified (UHPLC-MS/MS). Ferreira et al. evaluated samples collected in the autumn, winter and spring, seasons with different environmental factors such as temperature, ultraviolet radiation, rain index and soil nutrients. No significant correlations between the meteorological data and the biological potential were observed, demonstrating that the species studied was well adapted to the different environmental conditions targeted in the study.

An important aspect linked to the use of plants, medicinal or not, was contextualized in the work of Silva et al. (2022), who studied the peels and seeds of avocado (*Persea americana*), which represent 30% of the fruit and are usually discarded as waste, generating environmental problems and the loss of bioactive compounds that remain in the biomass after processing [4]. The authors showed that avocado residues retain several nutrients such as minerals (Ca, Mg, Mn and Zn) and high amounts of essential fatty acids such as linoleic, palmitic and oleic acids. The chemical profile obtained via paper spray mass spectrometry (PSMS) showed fifty-five metabolites, including phenolic compounds, hydroxycinnamic acids, flavonoids and alkaloids, in the residues. The ethanol extract of the peels was the best acetylcholinesterase inhibitor, with no significant difference (*p* > 0.05) compared to the control, eserine. The seed extracts exhibited an in vivo neuroprotective effect against rotenone-induced damage in an in vivo *Drosophylla melanogaster* model [4]. This work contributed some interesting points to the scope of research on natural products, such as a preoccupation with sustainability and a circular economy, the choice of environmentally favorable solvents for extraction and the role of complementarity in vivo assays. Although toxicity, bioavailability and suitable formulations should be further investigated before using avocado residues in pharmacotherapy, this work showed the potential use of avocado residues as a bioresource in the development of low-cost drugs and functional foods with neuroprotective effects.

Bacteria and fungi are also important producers of bioactive metabolites. The purification of a catechol-type siderophore from *Streptomyces tricolor* was studied with the aim of inducing recovery from iron-deficiency-induced anemia in in vivo rat model (Barakat et al., 2022) [11]. Siderophores are low-molecular-weight natural compounds secreted by microorganisms that act as iron chelators. Due to this characteristic, siderophores have useful therapeutic applications, especially in iron-overload diseases (hemosiderosis, β-thalassemia, hemochromatosis and accidental iron poisoning). Iron chelators are also useful in cancer therapy because cancer cells have higher requirements of iron when compared to healthy cells. Barakat et al. (2022) focused on applications related to the damages caused by irondeficiency-induced anemia, showing that sidereophores improved weight gain and were effective during recovery from anemia [11]. The results led the authors to propose some hypotheses and created opportunities to delineate the detailed mechanism of the changes observed and elucidate iron pathways.

*Lacticaseibacillus rhamnosus* XN2, a bacteriocin-producing strain isolated from the naturally fermented yak yoghurt produced in Xining and Qinghai Provinces in China, was the target of the work of Wei et al. (2022) [12]. The source of the bacteria studied, yogurt from yak milk farmed in the Himalayan region, exemplifies the vastness of the area of natural products and the local value of the studies, as well as the global importance of the results in expanding knowledge in the area of biodiversity and ecosystem services. Bacteriocins from lactic acid bacteria are peptides secreted by some lactic acid bacteria with natural antimicrobial activities against other microorganisms, including food spoilage and pathogens. *L. rhamnosus* XN2 demonstrated antibacterial activity against *Bacillus subtilis*, *B. cereus*, *Micrococcus luteus*, *Brochothrix thermosphacta*, *Clostridium butyricum*, *S. aureus*, *Listeria innocua*, *L. monocytogenes* and *Escherichia coli.* Semi-purified, cell-free supernatants of *L. rhamnosus* XN2 showed bactericidal activity, probably due to the disruption of the sensitive bacteria membrane, as suggested by a flow cytometry analysis. The production of α-haemolysin and biofilm formation were observed for sub-lethal concentrations of the semi-purified material. Bacteriocin was further purified via reversed-phase high-performance liquid chromatography (RP-HPLC), and its amino acid sequence was determined to be Met-Lue-Lys-Lys-Phe-Ser-Thr-Ala-Tyr-Val [12]. The authors concluded that *L. rhamnosus* XN2 and its bacteriocin showed antagonistic activity at both cellular and quorum-sensing levels.

In relation to fungi, their secondary metabolites usually have diverse applications in the food, cosmetic, beverage and textile industries, and their biological activities enable their use in the development of new drugs. Some fungal pigments, such as azaphilones and isolated β-carotene, have already found commercial and industrial applications, and most of the fungi explored for the production of pigments are from a few genera such as *Monascus*, *Talaromyces*, *Aspergillus*, *Penicillium* and *Fusarium*. Lagashetti et al. (2022) focused on a less-common fungal species, isolated from the infected leaves of *Maytenus rothiana* and identified via morphological and molecular methods as *Gonatophragmium triuniae* [13]. Its growth under different conditions revealed the conditions suitable for pigment production, and biological screening demonstrated the antibacterial and antioxidant activities of *G. triuniae* extracts. The major orange-colored pigment produced by the species was identified as 1,2-dimethoxy-3 H-phenoxazin-3-one. The authors note that pigments and other bioactive secondary metabolites of *G. triuniae* have potential applications in the textile and pharmaceutical industries.

Fungi of the genus *Penicillium* are widely studied as sources of bioactive compounds, but research on these fungi and their metabolites is far from complete, as shown in the work of Cadelis et al. (2022) [14]. Studying P. *bissettii* and *P*. *glabrum*, this group isolated five known polyketide metabolites, penicillic acid, citromycetin, penialdin A, penialdin F and myxotrichin B. During the derivatization of penicillic acid, a novel dihydro derivative was produced, providing evidence for the existence of an open-chained γ-keto acid tautomer in the starting material. Penicillic acid and penialdin F were found to inhibit the growth of methicillin-resistant *S. aureus*, which is important in view of the clinical problems associated with resistant microbial strains, which mainly involve hospitalized patients. Two other metabolites, penialdin F and citromycetin, were active against *Mycobacterium abscessus* and *M. marinum* [14].

This Special Issue also received three interesting review contributions. The first of these addressed recently discovered secondary metabolites from *Streptomyces* species and was prepared by Lacey and Rutledge [15]. Their review, with a particular focus on the year 2020, presented 74 novel secondary metabolites from *Streptomyces* species, with a wide range of chemical scaffold variability, including the cyclic peptides ulleungamide, viennamycins A and B and pentaminomycins C–E, metabolites with complex chemical

structures. Linear peptides such as spongiicolazolicins A and B were isolated from marine species, and linear polyketides (e.g., adipostatins E–J, trichostatic acid B, trichostatin A and chresdihydrochalcone), terpenoids (e.g., napyradiomycins and flaviogeranins), polyaromatics (e.g., baikalomycins A–C and gardenomycins A and B), macrocycles (e.g., conglobatins and somamycins) and furans (e.g., furamycins) are among the compounds discussed in the review [16]. The authors noted that the *Streptomyces* species reported in their review were isolated from a wide range of environments, possess a diversity of novel chemical structures and represent a thriving and multifaceted area of drug discovery research.

The second review contribution focused on <sup>13</sup>C-NMR data from 504 pentacyclic triterpenoids isolated from plants of the Celastraceae family, covering the period of 2001 to 2021 [15]. This class of secondary metabolites is of the utmost importance as they are reported to possess varied biological potential as antiviral, antimicrobial, analgesic, antiinflammatory and cytotoxic agents against various tumor cell lines. The review covered the pentacyclic triterpenoids of friedelane, quinonemethide, aromatic, dimer, lupane, oleanane and ursane, among other classes. The data reported by Camargo et al. (2022) highlighted the amazing structural diversity of pentacyclic triterpenes and the complexity of some representatives, such as the dimeric molecules [15]. The <sup>13</sup>C-NMR data presented in this review are an enormous contribution to the structural elucidation of new compounds of this class of terpenes.

Last, but not least, Amen and colleagues from Prof. Shimizu's group reviewed the sources, bioactivities, biosynthesis and spectroscopic features of naturally occurring chromone glycosides. The compounds addressed in the review were described from plants (angiosperms) of thirty-three families, three families of ferns, four species of lichens, three species of fungi and three families of actinobacteria [17]. O-glycosides or C-glycosides were analyzed separately; phenyl and isoprenyl chromone glycosides and phenyl ethyl chromone glycosides were then analyzed, followed by the class of chromone glycosides with additional heterocyclic moieties. Hybrids of chromones with other classes of secondary metabolites, hybrids of furano-chromones with cycloartane triterpenes and hybrids of chromones with secoiridoids were presented in sequence, and finally, representants of the groups of chromone alkaloids and aminoglycosides, were discussed. In the second part, the spectroscopic features of the chromones were carefully described, including UV, IR, <sup>1</sup>H and <sup>13</sup>C-NMR data of the 192 chromones [17]. This is indeed a great collaboration for the prompt identification of chromone metabolites and an incentive to conduct deeper studies with this class of metabolites.

In view of the papers that comprise this Special Issue, we believe that our objectives have been achieved. We are thankful for all the contributions received, and hope that the papers will be of interest to all readers of *Molecules*. Finally, natural products still have much to contribute to humanity, and we wish great results and good discoveries to all authors and readers in their upcoming research.

**Funding:** We acknowledge the grants from CNPq 31150/2022 and FAPEMIG 00255-18.

**Conflicts of Interest:** The author declares no conflict of interest.

### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

## *Article Bauhinia forficata* **Link Infusions: Chemical and Bioactivity of Volatile and Non-Volatile Fractions**

**Eliane Przytyk Jung <sup>1</sup> , Beatriz Pereira de Freitas <sup>2</sup> , Claudete Norie Kunigami <sup>1</sup> , Davyson de Lima Moreira 3,\* , Natália Guimarães de Figueiredo <sup>4</sup> , Leilson de Oliveira Ribeiro 1,\* and Ricardo Felipe Alves Moreira <sup>5</sup>**


**Abstract:** This study aimed to evaluate *Bauhinia forficata* infusions prepared using samples available in Rio de Janeiro, Brazil. As such, infusions at 5% (*w*/*v*) of different brands and batches commercialized in the city (CS1, CS2, CS3, and CS4) and samples of plant material botanically identified (BS) were evaluated to determine their total phenolic and flavonoid contents (TPC and TFC), antioxidant capacity (ABTS•<sup>+</sup> , DPPH• , and FRAP assays), phytochemical profile, volatile compounds, and inhibitory effects against the α-amylase enzyme. The results showed that infusions prepared using BS samples had lower TPC, TFC and antioxidant potential than the commercial samples (*p* < 0.05). The batch averages presented high standard deviations mainly for the commercial samples, corroborating sample heterogeneity. Sample volatile fractions were mainly composed of terpenes (40 compounds identified). In the non-volatile fraction, 20 compounds were identified, with emphasis on the CS3 sample, which comprised most of the compounds, mainly flavonoid derivatives. PCA analysis demonstrated more chemical diversity in non-volatile than volatile compounds. The samples also inhibited the α-amylase enzyme (IC<sup>50</sup> value: 0.235–0.801 mg RE/mL). Despite the differences observed in this work, *B. forficata* is recognized as a source of bioactive compounds that can increase the intake of antioxidant compounds by the population.

**Keywords:** "pata-de-vaca"; phytochemical profile; bioactive compounds; antioxidant capacity; α-amylase inhibition; SPME technique

**1. Introduction**

*Bauhinia* is a genus comprising over 300 species widely distributed in tropical and subtropical forests. In Brazil, 64 species belonging to the Fabaceae family were identified and are commonly known as "pata-de-vaca" due to the shape of their leaves. Most species are of Asian origin; however, *Bauhinia longifolia* (Bong.) Steud. and *Bauhinia forficata* Link are native species from Brazil [1,2].

*B. forficata* is widely used in Brazilian folk medicine due to its beneficial effects on different diseases and human disorders such as rheumatism, local pain, uric acid, and uterine problems [3], but it is primarily used to treat type II diabetes [4]. The beneficial effects are associated with various biocompounds present in *B. forficata*, such as flavonoids, alkaloids, and terpenes/terpenoids [2,5]. The flavonoid compounds are highlighted since they are the major class in *B. forficata* extracts. Farag et al. [6] registered the presence of quercetin and kaempferol derivatives in different species of the *Bauhinia* genus, including *B. forficata*.

**Citation:** Jung, E.P.; de Freitas, B.P.; Kunigami, C.N.; Moreira, D.d.L.; de Figueiredo, N.G.; Ribeiro, L.d.O.; Moreira, R.F.A. *Bauhinia forficata* Link Infusions: Chemical and Bioactivity of Volatile and Non-Volatile Fractions. *Molecules* **2022**, *27*, 5415. https:// doi.org/10.3390/molecules27175415

Academic Editor: Jacqueline Aparecida Takahashi

Received: 20 July 2022 Accepted: 20 August 2022 Published: 24 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In Brazil, *B. forficata* is mainly commercialized dried and used to prepare infusions. Thus, under Brazilian law, the *Bauhinia* tea is associated to food products, so it is not mandatory to indicate the content of bioactive or toxic compounds, as in a limited manner in herbal products [7]. *B. forficata* infusions were used in different in vivo studies, such as that reported by Salgueiro et al. [8], who evaluated the effects of infusions on oxidative stress, liver damage, and glycemia in mice. Nevertheless, data on the content of bioactive compounds, antioxidant capacity, and volatile compounds, among other parameters of this plant, to compare botanically identified and commercialized samples and their infusions are scarce in the literature. Since it is well known that various factors such as climate, processing, and storage conditions may influence the content of bioactive compounds and the volatile fraction of medicinal plants [9,10], there is a clear need for further studies.

Despite that, to date, there are no data available on the volatile composition of *B. forficata* infusions. This fraction cannot be underestimated since *B. forficata* is prepared by infusion or decoction and, therefore, some of the volatile content may disperse in the beverage (hydrolate) and contribute to its beneficial actions besides the aroma. This approach has already been evaluated for other medicinal plants, and the migration of terpenoid and other compounds classes present in the essential oil of the plant for infusion was observed [11,12].

For such an evaluation, headspace solid-phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME/GC–MS) has been reported as a fast, sensitive, and solvent-free technique for analyzing the extraction and isolation of volatile and semi-volatile compounds, and it has been widely used since its invention in 1989 [13,14]. Furthermore, interference from the infusion matrix may be drastically reduced while the headspace analytes are trapped in the fiber [15]. Thus, this technique has been successfully applied to analyze volatile compounds in infusions and teas [16,17].

In this sense, this work aimed to perform a comprehensive chemical characterization of the volatile and non-volatile fractions of botanically identified and commercial samples of *B. forficata* used to prepare infusions at 5%. The antioxidant capacity measured by ABTS•**<sup>+</sup>** , DPPH• and FRAP assays and inhibitory activity of α-amylase of the samples were also determined.

#### **2. Results**

#### *2.1. Bioactive Compounds and Antioxidant Capacity of B. forficata Infusions*

The TPC, TFC and antioxidant capacity of the *B. forficata* infusions are summarized in Table 1. It should be pointed out that the results presented in this study for TPC and TFC are expressed as rutin equivalents (RE) since this compound belongs to the flavonoids class, which is the major class in this species [6]. The values of TPC varied from 1923 to 6355 mg RE/100 g. Compared to the literature, the highest value found in this study, which was for the dry basis (7222 mg RE/100 g), is superior to that reported by Port's et al. [18], who evaluated different infusions of herbs from the Brazilian Amazonian region. Even though these authors did not evaluate *B. forficata*. However, their approach was the closest to this study, reporting results of the chemical evaluation for a *B. ungulata* infusion at 2% (g/mL) (2367 mg GAE/100 g dry basis). By calculation, at 5%, 5918 mg GAE/100 g dry basis would be found. Comparisons with data from the literature are difficult since few studies used the same species, and even when the species were the same, the results were expressed using different chemical standards, as in the example above. Additionally, it is easier to find data on *B. forficata* extracted with organic solvent than with hot water (infusion). Thus, our discussion will be focused on the differences observed among the brands and respective batches evaluated herein.


**Table 1.** Total phenolic content (TPC), total flavonoid content (TFC) and antioxidant capacity of *B. forficata* infusions.

Abbreviations in the "Samples" column represent the different batches of each one of the brands evaluated. Different lowercase letters in the same column indicate that the results are statistically different (*p* < 0.05). Different uppercase letters in the same column indicate a statistically significant difference among groups (BS, CS1, CS2, CS3 and CS4) (*p* < 0.05). <sup>1</sup> Results expressed as mg RE/ 100 g. <sup>2</sup> Results expressed as µmol Trolox/g. <sup>3</sup> Results expressed as µmol Fe2+/g. BSB1 = botanical sample batch 1; BSB2 = botanical sample batch 2; BSB3 = botanical sample batch 3; CS1B1 = commercial sample 1 batch 1; CS1B2=commercial sample 1 batch 2; CS2B1 = commercial sample 2 batch 1; CS2B2 = commercial sample 2 batch 2; CS2B3 = commercial sample 2 batch 3; CS3B1 = commercial sample 3 batch 1; CS3B2 = commercial sample 3 batch 2; CS3B3 = commercial sample 3 batch 3; CS4B1 = commercial sample 4 batch 1; CS4B2 = commercial sample 4 batch 2; CS4B3 = commercial sample 4 batch 3. Results as the mean ± standard deviation (triplicate).

The values for TPC, TFC, and antioxidant capacity measured by DPPH• , ABTS•<sup>+</sup> , and FRAP assays varied from 1923 to 6355 mg RE/100 g, 482 to 3700 mg RE/100 g, 19 to 206 µmol Trolox/g, 27 to 204 µmol Trolox/g, and 85 to 644 µmol Fe2+/g, respectively. This corroborates that variations among samples and batches were high (Table 1). Among batches of the commercial samples, the highest values for TPC, TFC and antioxidant capacity (CS4B2 and CS3B3) were observed. These were higher than values reported to botanically identified sample (BS), which may be explained by differences in cultivation practices and the way the plants were processed. For example, the drying time may increase the degradation of plant bioactive compounds, whereas soil characteristics and precipitation conditions may affect the biosynthesis of secondary metabolites [9,10].

CS4B2 presented the highest TPC and FRAP values. For the TFC and DPPH• and ABTS•**<sup>+</sup>** assays, CS3B3 presented the highest values (Table 1). The literature points to a direct relationship between TPC and antioxidant capacity; however, in this study, the sample that presented the highest TPC did not show the highest values for antioxidant capacity measured by all assays employed. This corroborates that the phytochemical composition of plant extracts may interact differently with radical species, which helps explain the results found.

High standard deviations were observed in CS2 and CS4 samples. The variation coefficient for the TFC reached 75% in CS2, for example, confirming the heterogeneity among the sample batches. The low standard deviation of the BS may be associated mainly with the standardization of the processing, which was followed from the harvest of leaves to drying. In addition, the harvest was from the same tree, although it took place in different seasons. This may also justify the low standard deviation of CS1 and CS3. Furthermore, conditions such as storage time, temperature, and kind of package have influence on the stability of bioactive compounds.

Since the samples showed heterogeneous batches according to the statistical analysis for this set of experiments, two groups were observed from their averages: one composed of the BS, CS1, CS2, and CS4 groups, for which no statistically significant differences were observed for the TPC, TFC, DPPH• , ABTS•**<sup>+</sup>** , and FRAP assays (*p* > 0.05), and the other represented by CS3 alone. These data provide important information about the production chain of *B. forficata*, rendering evident the need to standardize the steps that involve from harvest to distribution to deliver to consumers a product that guarantees its bioactive properties. *B. forficata* is widely used in Brazilian folk medicine due to its beneficial effects for treating rheumatism, local pain, uric acid, uterine problems [3], and, especially, type II diabetes [4]. This is possible due to the phytochemical profile of *B. forficata*, which is mainly composed of flavonoids, recognized for their antioxidant capacity [19].

#### *2.2. LC-HRMS Analysis*

A total of 20 phenolic compounds (Table 2), among flavonoids, phenolic acids, and other phenolic compounds, were tentatively identified in the samples. The majority are kaempferol and quercetin derivatives. The samples comprised flavonoid *O*-glycosides, thus in accordance with previously reported results, which prove its pharmacological action [20,21]. Additionally, polar compounds were identified in the samples in accordance with the polarity of the infusions.

**Compounds** *<sup>m</sup>***/***<sup>z</sup>* **[M–H]**<sup>−</sup> **exp. MS<sup>2</sup> Molecular Formula [M–H]**− **Samples BS CS1 CS2 CS3 CS4** 1 Caffeoyl tartarate 311.0401 179; 135 C13H11O<sup>9</sup> + 2 *Epi*-Catechin 289.0718 245; 203 C15H13O<sup>6</sup> + 3 Galloyl hexose 331.0670 169; 125 C13H15O<sup>10</sup> + + + + 4 Hydroxibenzoic acid 137.0244 - C7H5O<sup>3</sup> + + + + 5 Dihydroxibenzoic acid hexoside 315.0719 108; 152 C13H15O<sup>9</sup> + + 6 3-Caffeoyl quinic acid 353.0875 191 C16H17O<sup>9</sup> + + + + 7 Kaempferol 3-*O*-rhamnosyl-rutinoside 739.2136 284 C33H39O<sup>19</sup> + 8 Rutin 609.1468 300 C27H29O<sup>16</sup> + + + + + 9 Myricitrin 463.0880 316 C21H29O<sup>12</sup> + <sup>10</sup> Quercetin 3-*O*-glucopyranoside (Isoquercetin) 463.0917 301; 300 C21H29O<sup>12</sup> + + + + + <sup>11</sup> Quercetin-*O*-pentoside (Quercetin-*O*-arabinoside) 433.0780 300; 301 C20H17O<sup>11</sup> + + + + + 12 Quercetin 3-*O*-rhamnoside 447.0933 284; 285 C21H29O<sup>11</sup> + + + + + 13 Kaempferol 3-*O*-glucoside 447.0975 - C21H29O<sup>11</sup> + + + + + 14 Kaempferol 3-*O*-rutinoside 593.1533 327; 284; 285 C27H29O<sup>15</sup> + + + + 15 Isorhamnetin 315.0502 300 C16H11O<sup>7</sup> + + + + + 16 Isorhamnetin 3-*O*-rutinoside 623.1638 300; 315 C28H31O<sup>16</sup> + 17 Quercetin 3-*O*-rhamnosyl-rutinoside 755.2087 300; 489 C33H39O<sup>20</sup> + + 18 Isorhamnetin 3-*O*-rhamnosyl-rutinoside 769.2201 605; 315 C34H41O<sup>20</sup> + + 19 Kaempferol 3-*O*-dirhamnoside 577.1595 431, 285, 284 C27H29O<sup>14</sup> + 20 Kaempferol-*O*-pentoside 417.0833 285, 284, 255, 227 C20H17O<sup>10</sup> +

**Table 2.** Tentatively identified compounds of *B. forficata* infusions.

BS: botanic sample; CS1: commercial sample 1; CS2: commercial sample 2; CS3: commercial sample 3; CS4: commercial sample 4. *m*/*z*—mass to charge ratio; MS2—fragments of the second stage of mass spectrometry.

Most phenolic compounds identified in this study were free phenolic compounds, esterified with sugars or other compounds with low molecular masses, such as quercetin 3-*O*-rhamnoside, Kaempferol 3-*O*-glucoside, and Isorhamnetin.

Rutin, Isoquercetin, Quercetin-*O*-pentoside, Quercetin 3-*O*-rhamnoside, Kaempferol 3-*O*-glucoside, Kaempferol 3-*O*-rutinoside and Isorhamnetin were the compounds detected in all samples. CS3 is the infusion with the greatest number of compounds that vary according to the batch. In CS1, CS2, and CS4, the same 11 flavonoids were identified with differences in the relative abundance of the ions. Compound 3 showed a precursor ion [M–H]¯ at 331.0670 *m*/*z* and a typical loss of a hexose in MS<sup>2</sup> resulting in a [M–H]¯

*m*/*z* 169 fragment. It was assigned as galloyl hexose. Compound 4 was assigned as hydroxibenzoic acid based on precursor ion [M–H]¯ at 137.0244 *m*/*z* and a very low error between experimental and theoretical mass of 0.1 ppm [22]. Compound 6 showed a precursor ion [M–H]¯ at 353.0875 *m*/*z* and the quinic acid fragment in MS<sup>2</sup> at *m*/*z* 191, been identified as 3-Caffeoyl quinic acid. Compound 10 was assigned as Quercetin 3-*O*-glucopyranoside by comparison with literature records (1 ppm error ([M–H]¯ *m*/*z* 463.0878) [23]. Compound 19 was assigned as Isorhamnetin 3-*O*-rhamnosyl-rutinoside based on precursor ion [M–H]¯ *m*/*z* 769.2190 and based in the loss of Isorhamnetin fragment at *m*/*z* 315. Kaempferol fragment ion at *m*/*z* 284 was used to identify compound 20 as Kaempferol 3-*O*-dirhamnoside along with the precursor ion [M–H]¯ *m*/*z* 577.1595 [6]. Identification of the other listed compounds by fragmentation data and exact mass were previously described by the authors [24,25].

The UPLC-ESI-Q-TOF MS/MS chromatographic technique was an efficient tool to characterize and identify the phenolic compounds in *B. forficata* infusions. It is important to highlight that the advantage of this technique is that, although it is not quantitative, one may relatively quantify the compounds, even the isomeric forms (e.g., Catechin, *Epi*-catechin, and Quercetin-*O*-pentoside), and, in case of a lack of standards, the compound assignments may be made by comparison of UV spectra and MS data (accurate mass and fragmentation) with previous literature reports [6,22,23].

A PCA analysis of the non-volatile chemical composition showed three distinct groups: **I**—CS3B2, CS3B3 and CS4B2; **II**—CS1B1, CS1B2, CS2B1, CS2B2, CS4B1, CS4B3 and BSB1; **III**—CS3B1, BSB2 (Figure 1A). These results demonstrate great chemical variability between the different samples, although flavonoids Rutin, Isoquercetin, Quercetin-*O*-pentoside, Quercetin 3-*O*-rhamnoside, Kaempferol 3-*O*-glucoside, Kaempferol 3-*O*-rutinoside and Isorhamnetin were detected in all samples. *Molecules* **2022**, *27*, x FOR PEER REVIEW 6 of 15

**Figure 1.** Principal component analysis of (**A**) non-volatile compounds and (**B**) volatile compounds. BS: botanic sample; CS1: commercial sample 1; CS2: commercial sample 2; CS3: commercial sample 3; CS4: commercial sample 4. B is relative to the batch. \*I—BSB3 (rich in Caryophyllene oxide), CS1B2, CS3B2 and CS4B2 (rich in Spathulenol); \*II—CS2B1 (rich in 2-Propyl-1-heptanol). **Figure 1.** Principal component analysis of (**A**) non-volatile compounds and (**B**) volatile compounds. BS: botanic sample; CS1: commercial sample 1; CS2: commercial sample 2; CS3: commercial sample 3; CS4: commercial sample 4. B is relative to the batch. \*I—BSB3 (rich in Caryophyllene oxide), CS1B2, CS3B2 and CS4B2 (rich in Spathulenol); \*II—CS2B1 (rich in 2-Propyl-1-heptanol).

#### *2.3. HS-SPME/CG–MS 2.3. HS-SPME/CG–MS*

sition of CS2.

centage (%).

**(min) LRI (a) Compound Chemical** 

**Rt** 

The identification and relative concentrations of the volatile compounds in the five herbal infusions of *B. forficata* are shown in Table 3, in order of retention time (Rt), and increasing Linear Retention Index (LRI). Forty volatile compounds were tentatively iden-The identification and relative concentrations of the volatile compounds in the five herbal infusions of *B. forficata* are shown in Table 3, in order of retention time (Rt), and increasing Linear Retention Index (LRI). Forty volatile compounds were tentatively identified, of which

tified, of which only seven were detected in all samples: 2-Propyl-heptanol (7.69–19.42%), Geranyl acetone (4.38–7.31%), Dodecanol (3.11–11.37%), β-Ionone (0.71–5.84%), Spathulenol (11.78–30.87%), Caryophyllene oxide (2.76–17.46%), and Benzoic acid 2-ethylhexyl

isoprenoids, sesquiterpenes, and monoterpenes, as well as hydrocarbons, alcohols, esters, aldehydes, ketones, and acids. Among all chemical groups found in the volatiles of the *B. forficata* infusions, sesquiterpenes (hydrocarbon and oxygenated) were present in a higher number (17) and represented most of the composition of the BS (63%), CS1 (61%), CS3 (50%), and CS4 (53%). Esters (31%) and alcohols (29%) accounted for most of the compo-

**Table 3.** Tentatively identified compounds of *B. forficata* infusions with their respective relative per-

**Class BSB3 CS1B2 CS2B1 CS3B2 CS4B2** 

14.00 1185 1-Decanal A 0.10 ± 0.04 - 0.57 ± 0.22 - - 14.30 1193 2-Propyl-1-heptanol AL 3.35 ± 0.35 7.69 ± 0.62 19.42 ± 2.52 9.66 ± 4.93 8.96 ± 3.07 16.40 1195 Estragole PP - 0.30 ± 0.00 - - 0.44 ± 0.21 18.44 1357 Eugenol PP 0.24 ± 0.00 - - - - 20.10 1428 β-Caryophyllene S 0.85 ± 0.10 - - - - 20.30 1429 α-Ionone N 3.59 ± 0.47 1.55 ± 0.08 1.64 ± 0.04 - - 20.90 1448 Geranyl acetone N 6.88 ± 1.08 7.31 ± 0.00 5.18 ± 0.76 5.02 ± 1.08 4.38 ± 1.25 20.92 1452 α-Humulene S 1.22 ± 0.45 - - - - 21.00 1461 Alloaromadendrene S 0.70 ± 0.03 - - - - 21.20 1472 *p*-Benzoquinone K - 0.66 ± 0.04 1.50 ± 0.22 0.99 ± 0.03 - 21.40 1480 Dodecanol AL 4.00 ± 3.75 3.11 ± 0.51 7.14 ± 1.39 3.94 ± 1.27 8.37 ± 0.01 21.70 1485 Deydro-β-ionone N - - 5.30 ± 0.50 - 1.17 ± 0.36 21.80 1486 β-Ionone N 4.24 ± 0.05 3.08 ± 0.11 0.71 ± 0.23 2.54 ± 0.38 5.84 ± 1.19 only seven were detected in all samples: 2-Propyl-heptanol (7.69–19.42%), Geranyl acetone (4.38–7.31%), Dodecanol (3.11–11.37%), β-Ionone (0.71–5.84%), Spathulenol (11.78–30.87%), Caryophyllene oxide (2.76–17.46%), and Benzoic acid 2-ethylhexyl ester (1.12–20.14%). The volatile compounds included terpenoids, represented by C13-norisoprenoids, sesquiterpenes, and monoterpenes, as well as hydrocarbons, alcohols, esters, aldehydes, ketones, and acids. Among all chemical groups found in the volatiles of the *B. forficata* infusions, sesquiterpenes (hydrocarbon and oxygenated) were present in a higher number (17) and represented most of the composition of the BS (63%), CS1 (61%), CS3 (50%), and CS4 (53%). Esters (31%) and alcohols (29%) accounted for most of the composition of CS2.


**Table 3.** Tentatively identified compounds of *B. forficata* infusions with their respective relative percentage (%).

(a) Linear Retention Index (LRI) calculated for all components using a homologous series of *n*-alkanes analyzed under the same conditions as the samples; (-) not detected. A—aldehyde, AL—alcohol, PP—phenylpropanoid, S—sesquiterpene, N—norisoprenoid, K—ketone, OM—oxygenated monoterpene, OS—oxygenated sesquiterpene, CA—carboxylic acid, HC—hydrocarbon, E—ester, and PH—phenol. BSB3 = botanically identified sample, batch 3; CS1B2 = commercial sample brand 1, batch 2; CS2B1 = commercial sample brand 2, batch 1; CS3B2 = commercial sample brand 3, batch 2; CS4B2 = commercial sample brand 4, batch 2. Relative percentage as the mean ± standard deviation (duplicate).

There are no data in the literature on the volatile composition of *B. forficata* infusions or any species of the *Bauhinia* genus. However, there are two studies that identified constituents of essential oils of this species and demonstrated that they are essentially composed of sesquiterpenoids. Duarte-Almeida et al. [26] and Sartorilli and Correa [27] evaluated the composition of essential oils in *B. forficata* and reported that the content of sesquiterpenoids was 87% and 96%, respectively. Our results and those from essential oils [26,27] are a great evidence that a mostly sesquiterpenic volatile fraction composition may be characteristic of this species.

It is well established that many sesquiterpenes and their alcohol, aldehyde, and ketone derivatives are biologically active or precursors of metabolites with biological functions, while others have desirable fragrance and flavoring properties [28]. Spathulenol (8.53–25.86%) and Caryophyllene oxide (2.76–17.46%) were two of the major compounds in all samples. Both compounds are known to possess several biological activities. Nascimento et al. [29] demonstrated antioxidant, anti-inflammatory, antiproliferative, and antimycobacterial activities of spathulenol, and a moldy and herbaceous odor is attributed to this compound [30]. In turn, Caryophyllene oxide has a floral and woody odor [31,32], and biological activities such as anticholinesterase, analgesic, anti-inflammatory, and antifungal activities were also reported [33,34]. Regarding the class of norisoprenoids (C13), they were detected in all samples at concentrations ranging from 7.56% to 14.71%, highlighting Geranyl acetone and β-Ionone. It is reported that they present a significant aromatic impact in fruits such as grapes, apples, lychee, and mango [35,36], with a floral odor being attributed to them [37].

Attention is drawn to the identification of Bisphenol A (BPA) and Dibutyl phthalate (DBP) in some samples evaluated here, especially CS2, which showed important concentrations of these contaminants in its volatile fraction (9.24% and 3.82%, respectively). As any agricultural product, these herbs may be subjected to chemical contaminations due to agricultural practices, especially in stages when a plastic material is used as packaging or support or due to soil treatment, cultivation in contaminated soil, and other factors [38,39]. Furthermore, the migration of these plasticizers that constitute the packaging cannot be ruled out since it is known that this is the main source of exposure to this type of contaminant [39]. Di Bella et al. [39] and Lo Turco et al. [40] evaluated the BPA contamination of spices and herbs from different origins and found it to be present in several samples. Despite concluding that the ingestion of these contaminants does not imply a risk to human health, one cannot disregard their existence, and mechanisms to mitigate them must be evaluated, such as proposing other packaging materials free from them.

In general, the observed differences among the volatile fraction patterns of the infusions were lower than those observed for non-volatile (Figure 1B). Only CS2B2 formed another group by PCA analysis (Figure 1B). Indeed, different origins of the samples with their unique ecological settings as well as features intrinsic to the medicinal herbs may explain this difference [12]. Moreover, Arsenijevi´c et al. [12] stressed that compounds present in the volatile fraction of infusions play an important role in the antioxidant capacity of these products, thus rendering this evaluation relevant, although it was still not possible to measure it in this work. Once again, we highlight that the results obtained herein are the first step towards revealing the beneficial health effects of *B. forficata* infusions through chemical diversity after evaluating their non-volatile and volatile fractions.

#### *2.4. Assay for α-Amylase Inhibition*

In this set of experiments the effect of *B. forficata* infusions that presented better results for TPC, TFC and antioxidant capacity was investigated. The results revealed that all infusions inhibited the α-amylase activity. Based on the IC<sup>50</sup> values, which represent the concentration required to inhibit 50% of the enzyme activity, the CS2B1 sample was the one that showed the greatest potential for enzyme inhibition, as it showed the lowest IC<sup>50</sup> value (0.235 mg RE/mL). The IC<sup>50</sup> values were 0.235 mg RE/mL, 0.245 mg RE/mL, 0.287 mg RE/mL, 0.489 mg RE/mL, and 0.801 mg RE/mL for CS2B1, CS4B2, CS1B2, BSB3, and CS3B2, respectively. Even though CS4B2 presented the highest TPC, this sample exhibited a higher IC<sup>50</sup> value. It is suggested that the inhibition of α-amylase activity may be due to other phytochemicals also present in the infusions such as terpenoids, which were detected in the samples by HS-SPME/CG–MS. However, it is well known that phenolic compounds, mainly flavonoids, are excellent inhibitors of digestive enzymes. Flavonoids and their derivatives have the ability to reduce the potency of α-amylase and α-glucosidase by either interacting with or inhibiting specific positions of the enzyme [41]. However, other classes of compounds should not be neglected as published by Papoutsis et al. [42], which reported in their review the positive effects of terpenoids, carotenoids, among others compounds on inhibition of α-amylase activity. It is important to note that these compounds should be bioavailable after digestion to act on digestive enzymes. Thus, future studies on this subject should be addressed.

Acarbose is widely used in medicine as an inhibitor of digestive enzymes related to the breakout of polysaccharides. As these enzymes are inhibited, there is a reduction in glucose absorption and, consequently, a decrease in the postprandial blood glucose level elevation, which helps reduce the risk of Diabetes mellitus, for example [42]. Its IC<sup>50</sup> value was found to be 0.034 mg/mL. Thus, a lower concentration of this substance is required to inhibit 50% of the α-amylase activity when compared to *B. forficata* infusions. However, it should be noted that this medicinal plant is widely used in folk medicine as an adjuvant in treating hyperglycemia by the population, especially those in vulnerable conditions [43].

It is important to demonstrate that infusions prepared from commercially available herbs showed an important inhibitory action on the enzyme despite being less potent than acarbose. Furthermore, cytotoxicity was not observed when different fractions from *B. forficata* were evaluated by Franco et al. [44]. These facts reinforce the biological and pharmacological potential of *B. forficata* as hypoglycemiant agent, which has an important role in Brazilian folk medicine, primarily because it is abundant and easily accessible.

#### **3. Material and Methods**

#### *3.1. Plant Material*

*B. forficata* leaves were collected in Petropolis, Rio de Janeiro, Brazil (22◦30004.6300 S, 43◦070058.2000 W, altitude: 958 m) in different seasons (winter, spring, and summer-2018/2019). Voucher specimens were deposited at the Herbarium of the Department of Botany of the Federal University of Rio de Janeiro, under registration number RFA 40.615. The samples were dried in an oven with forced air circulation at 45 ◦C, then disintegrated in a domestic blender to obtain a powered material, which was used to prepare the infusions. These samples were named BSB1 (winter), BSB2 (spring), and BSB3 (summer).

Four commercial samples purchased from local markets in the city of Rio de Janeiro were also evaluated. Two batches of commercial sample 1 (CS1) and three batches of the other samples (CS2, CS3, and CS4) were acquired, resulting in samples CS1B1, CS1B2, CS2B1, CS2B2, CS2B3, CS3B1, CS3B2, CS3B3, CS4B1, CS4B2, and CS4B3, which were used to prepare the infusions.

### *3.2. Preparing the Infusions*

The infusions were prepared by adding 50 mL of boiling water to 2.5 g of the samples (5% *w*/*v*). After that, they were allowed rest at room temperature for 20 min. The extracts were filtered and transferred to a volumetric flask, in which the volume was quenched with distilled water until reaching 50 mL [45].

#### *3.3. Analysis*

#### 3.3.1. Total Phenolic Content (TPC)

The TPC analysis was performed using the Folin-Ciocalteu reagent (Imbralab, Ribeirão Preto, Brazil), following the method described by Singleton and Rossi [46]. For the reactions, 250 µL of the filtered and appropriately diluted extract was mixed with 1250 µL of 10% Folin-Ciocalteu reagent and 1000 µL of a 7.5% (*w*/*v*) sodium carbonate solution. Thereafter, the samples were heated at 50 ◦C for 15 min and cooled at room temperature. The absorbance was measured at 760 nm. A calibration curve was constructed using the rutin (Sigma-Aldrich, St Louis, MO, USA) standard with concentrations ranging from 16 mg/L to 166 mg/ L (linear regression: y = 0.0034x−0.0128; R<sup>2</sup> = 0.9988). The TPC is expressed as milligrams of rutin equivalent per 100 g (mg RE/100 g).

#### 3.3.2. Total Flavonoid Content (TFC)

The TFC was determined based on the method described by Zhishen et al. [47] with minor modifications. Here, 0.5 mL of extract was mixed with 3.2 mL of ultrapure water and 150 µL of NaNO<sup>2</sup> (5%, *w*/*v*). After homogenization, the mixture was left to rest for 5 min. Thereafter, 150 µL of AlCl<sup>3</sup> (10%, *w*/*v*) was added to the mixture, and 1 mL of NaOH (1 M) was added after 1 min. The absorbance was recorded at 510 nm with a spectrophotometer (Metash, Shanghai, China) using ultrapure water as a blank. The TFC was calculated using the calibration curve of rutin (Sigma-Aldrich, St. Louis, MO, USA) standard, with the concentration ranging from 99 mg/L to 595 mg/L (linear regression: y = 0.001x + 0.013; R <sup>2</sup> = 0.9974). The results are expressed as mg RE/100 g.

## 3.3.3. ABTS•<sup>+</sup> Assay

The antioxidant capacity was determined by the reduction of radical monocation, 2,20 azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•<sup>+</sup> ), according to the procedure described by Gião et al. [48]. The radical was obtained after the addition of 7 mmol/L of ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich, Saint Louis, MO, USA) to 2.45 mmol/L of a potassium persulfate solution (1:1 (*v*/*v*)). The mixture was left to react in the dark for 16 h. To obtain an absorbance of 0.700 ± 0.020 at 734 nm, the ABTS•<sup>+</sup> solution was diluted using ultrapure water. For the reactions, 30 µL of each filtered and diluted extract was mixed with 3000 µL of the ABTS•<sup>+</sup> solution. After 6 min, the absorbance was measured at 734 nm with a spectrophotometer (Metash, Shanghai, China) using ultrapure water as a blank. The ABTS•<sup>+</sup> antiradical activity was calculated using Trolox solutions (Sigma-Aldrich, Buchs, Switzerland) with different concentrations ranging from 240 to 2000 µmol (linear regression: y = 0.0003x + 0.0094; R <sup>2</sup> = 0.9989). The results are expressed as µmol of Trolox equivalents per gram (µmol TE/g).

#### 3.3.4. DPPH• Assay

The 2,20 -diphenyl-β-picrylhydrazyl radical (DPPH• ) (Sigma-Aldrich, Steinheim, Germany) scavenging activity of the extracts was determined according to the method described by Hidalgo et al. [49]. For the reactions, 100 µL of each diluted extract was added to 2900 µL of a DPPH• solution (6 <sup>×</sup> <sup>10</sup>−<sup>5</sup> M in methanol and diluted to an absorbance of 0.700 at 517 nm). The resulting solutions were allowed to stand for 30 min in the dark at room temperature. Then, the absorbance was measured at 517 nm with a spectrophotometer (Metash, Shanghai, China) using methanol as a blank. The DPPH• scavenging activity was calculated using Trolox solutions (Sigma-Aldrich, Buchs, Switzerland) with different concentrations ranging from 80 to 680 µmol (linear regression: y = 0.0008x + 0.017; R <sup>2</sup> = 0.9962). The results are expressed as µmol TE/g.

#### 3.3.5. FRAP Assay

The ferric reducing/ antioxidant power (FRAP) assay was performed according to the procedure reported by Benzie and Strain [50] with minor modifications. The stock solutions included 300 mM of an acetate buffer (pH 3.6), 10 mM of 2,4,6-tri(2-pyridyl)-striazine (Sigma-Aldrich, Buchs, Switzerland) in 40 mM of HCl, and 20 mM of FeCl3·6H2O. The working solution was prepared by mixing 25 mL of the acetate buffer, 2.5 mL of the TPTZ solution, and 2.5 mL of FeCl3·6H2O. Thereafter, 100 µL of each extract was reacted with 3000 µL of the working solution at 37 ◦C for 30 min, and the absorbance was measured at 593 nm. The FRAP activity was calculated using FeSO4·7H2O solutions with different concentrations ranging from 150 to 1200 µmol of Fe2+ (linear regression: y = 0.0008x + 0.0042; R<sup>2</sup> = 0.9992). The results are expressed as µmol of Fe2+ per gram (µmol Fe2+/g).

#### 3.3.6. LC-HRMS Analysis

The sample extract was dissolved in an aqueous solution containing formic acid (0.1%, *v*/*v*) and subjected to an ultra-performance liquid chromatography-quadrupole/time-of-flight

mass spectrometry (UPLCqTOF/MS; maXis Impact, Bruker Daltonics, Billerica, MA, USA) analysis. The separation was performed using a Hypersil C18 column (3 µm particle size, 2.1 mm × 150 mm). The column temperature was maintained at 40 ◦C. Subsequently, an aliquot of 20 µL was injected into the UPLC-ESI-qTOF system with a flow rate of 0.27 mL/min. The linear gradient elution of A (0.1% formic acid in water) and B (acetonitrile) was applied by employing the following method: 5% of B at the beginning; 5% to 9% of B for 5 min, 9% to 16% of B for 10 min, 16% to 36% of B for 18 min, 36% to 95% of B for 1 min, 95% of B for 12 min, 95% to 5% of B for 1 min, and 5% of B for 13 min. Data Analysis 4.2 software (Bruker Daltonics, Billerica, MA, USA) was used to interpret the data. The MS data were acquired in the negative mode using an electrospray ionization (ESI) source. The data were scanned for each test sample at a mass-to-charge ratio (*m*/*z*) from 50 to 1200. Highly pure nitrogen was used as the nebulizing gas and ultrahigh purity helium as the collision gas, and the capillary voltage was set at 5000 V. The ESI parameters included dry gas at 200 ◦C at a flow rate of 8 L/min and a nebulizer pressure of two bar [25].

### 3.3.7. HS-SPME/CG–MS

The infusions that presented better results for TPC, TFC and antioxidant capacity were subjected to an analysis of the volatile fraction by Headspace Solid-Phase microextraction followed by gas chromatography–mass spectrometry (HS-SPME/GC–MS).

The headspace volatiles analysis using SPME described by Wang et al. [51] was adopted with minor modifications. Volumes of 10 mL of freshly prepared infusions were placed into 20 mL clear glass vials and immediately capped and placed on a temperaturecontrolled water bath at 60 ◦C for 60 min with a SPME fiber coated with 100 µm of PDMS (100% polydimethylsiloxane; Supelco®, Bellefonte, PA, USA) pre-conditioned at 250 ◦C for 60 min and inserted into the headspace above the liquid surface. A system blank with an empty vial was run as a control assay. SPME fibers were desorbed at 250 ◦C for 5 min in the injection port of the chromatographic system described below.

The GC–MS analysis of the volatile fractions was carried out using an Agilent 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with an HP-5MS 5% phenylmethylsiloxane capillary column (30 m × 0.25 mm, 0.25 µm film thickness; Restek, Bellefonte, PA, USA) equipped with an Agilent 5975 mass selective detector in the electron impact mode (ionization energy: 70 eV) operating according to the following conditions. The oven temperature was initially maintained at 60 ◦C for one 1 min, then raised at the rate of 8 ◦C/min to 300 ◦C, staying at this temperature for 15 min. The injector and detector temperatures were set at 250 ◦C and 260 ◦C, respectively. The samples were injected in the splitless mode. A normalization technique was used to obtain quantitative data. Linear retention indices (LRI) were calculated for all components using a homologous series of *n*-alkanes (C7–C30, Sigma-Aldrich, Laramie, WY, USA) analyzed under the same conditions as the samples. The identification of the volatile fraction components was based on LRI relative to *n*-alkanes and computer matching with the Wiley275.L and Wiley7n.L libraries and comparisons of the fragmentation patterns of the mass spectra with published data [52].

#### 3.3.8. Assay for α-Amylase Inhibition

The infusions that presented better results for TPC, TFC and antioxidant capacity were subjected to the inhibition assay for α-amylase, performed as reported by Meng et al. [53] with minor modifications. Briefly, 100 µL of extract was mixed with an α-amylase solution (100 µL, 1.0 U/mL) (Sigma-Aldrich, St. Louis, MO, USA) in a phosphate buffer (pH 6.9) and 250 µL of a 1% starch solution. The incubation was carried out for 5 min at 37 ◦C. The enzyme reaction was stopped by adding dinitrosalicylic acid reagent (250 µL) (Sigma-Aldrich, Steinheim, Germany), and incubation was carried out for 15 min in boiling water. For the dilution, 2 mL of distilled water was added to the final reaction mixture. The absorbance was measured at 540 nm. The inhibitory effect was calculated according to Equation (1), where Abscontrol-1 results from the reaction without adding the enzyme,

which was replaced by the buffer solution, while the mixture of the enzyme and starch solution without extract was Abscontrol-2. The results were expressed as IC<sup>50</sup> (mg RE/mL). Acarbose (Supelco, Laramie, WY, USA) was used as a positive control to compare the inhibitory effects.

Inhibition percentage (%) = [1 − (Abssample−Abscontrol-1)/Abscontrol-2] × 100 (1)

#### *3.4. Statistical Analysis*

The data were statistically analyzed using Statistica software version 13 (Dell Inc., Tulsa, OK, USA), performing an analysis of variance (ANOVA) and Tukey's test to verify the differences among averages, considering the 95% confidence level. Experiments were performed in duplicate/triplicate, and the results are presented as the average ± standard deviation. Additionally, the principal component analysis (PCA) were used to assess the variance in the non-volatile and volatile samples. Results were processed using STATIS-TICA software version 10 (StartSoft Inc., Tulsa, OK, USA).

#### **4. Conclusions**

It is concluded that the samples presented different TPC, TFC and antioxidant potentials. The commercial CS4B2 and CS3B3 samples showed higher values for bioactive compounds and antioxidant capacity than botanically identified samples. However, both were mostly composed of flavonoid derivatives. PCA analysis demonstrated more chemical diversity in non-volatile than volatile compounds. This analysis may justify the differences observed in the results of the performed assays. To the best of our knowledge, this is the first time that volatile fraction obtained from *B. forficata* infusions has been carried out. It is very clear that it is an important fraction with regard to the aroma besides possible contribution to the biological properties. An inhibitory effect of all *B. forficata* infusions on the α-amylase enzyme was observed. Despite the differences reported in this work, *B. forficata* presents itself as a source of bioactive compounds that may increase the intake of antioxidant compounds by the population.

**Author Contributions:** Conceptualization, E.P.J., L.d.O.R. and R.F.A.M.; methodology, C.N.K., L.d.O.R. and E.P.J.; formal analysis, B.P.d.F., L.d.O.R. and N.G.d.F.; investigation, C.N.K., B.P.d.F., E.P.J., D.d.L.M. and L.d.O.R.; resources, L.d.O.R. and R.F.A.M.; data curation, C.N.K., L.d.O.R., D.d.L.M. and R.F.A.M.; writing—original draft preparation, E.P.J., L.d.O.R., N.G.d.F. and B.P.d.F.; writing—review and editing, D.d.L.M., L.d.O.R., N.G.d.F. and R.F.A.M.; supervision, L.d.O.R. and R.F.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within this article.

**Acknowledgments:** The authors acknowledge the support from the National Institute of Technology (INT), Federal University of Rio de Janeiro State (UNIRIO), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are available from the authors.

#### **References**


## *Article* **New Benzil and Isoflavone Derivatives with Cytotoxic and NO Production Inhibitory Activities from** *Placolobium vietnamense*

**Lien T. M. Do <sup>1</sup> , Tuyet T. N. Huynh 1,2 and Jirapast Sichaem 3,\***


**\*** Correspondence: jirapast@tu.ac.th; Tel.: +66-54237986

**Abstract:** The phytochemical investigation of *Placolobium vietnamense* stems led to the isolation of a new isoflavone derivative (**1**) and three new benzil derivatives (**2**–**4**), together with four known pyranoisoflavones (**5**–**8**). The structures of all isolated compounds were determined on the basis of extensive spectroscopic analyses, including NMR and HRMS spectral data, as well as comparison of their spectroscopic data with those reported in the literature. The cytotoxicity of all isolated compounds was assessed against the human liver hepatocellular carcinoma (Hep G2) cell line, and compound **1** displayed the most significant cytotoxicity with an IC<sup>50</sup> value of 8.0 µM. Furthermore, all isolated compounds were also tested for their inhibitory activity against NO production in RAW 264.7 macrophages. Of these, compound **1** exhibited the strongest inhibitory efficacy against the LPS-induced NO production with the IC<sup>50</sup> value of 13.7 µM.

**Keywords:** *Placolobium vietnamense*; placovinones A–D; benzil and isoflavone derivatives; cytotoxicity; NO production inhibition

## **1. Introduction**

*Placolobium* is a genus of plants in the family Fabaceae, which contains three accepted species. These are distributed throughout the world's tropical regions, some extending into temperate zones, especially in East Asia [1]. *Placolobium vietnamense* N.D.Khoi & Yakovlev is an indigenous plant species, known in Vietnam as 'Rang Rang'. It is a perennial tree with a straight, cylindrical trunk, and brown bark. The fruit is a small pod with a single seed. This plant is used as a folk remedy for snakebites, debility, and to increase strength after childbirth [1]. There has only been one investigation into the chemical constituents of *P. vietnamense* [1]. Previously, our group reported the isolation and structure elucidation of six isoflavonoids, including afrormosin, cladrastin, 8-*O*-methylretusin, millesianin C, barbigerone, and durallone from the EtOAc stem extract of this plant, together with their cytotoxicity. Encouraged by structurally diverse bioactive compounds from *Placolobium* species [2], the aim of this investigation is to revisit *P. vietnamense* in order to search for new bioactive compounds. We report herein the isolation and characterization of benzil and isoflavone derivatives from the stems of *P. vietnamense*. All isolated compounds were assessed for their cytotoxicity against human liver hepatocellular carcinoma (Hep G2) cell line, which is one of the most fatal cancers and has spread to the liver from other organs. Additionally, the inhibitory activity toward NO production in RAW 264.7 macrophages of all isolated compounds was also evaluated.

**Citation:** Do, L.T.M.; Huynh, T.T.N.; Sichaem, J. New Benzil and Isoflavone Derivatives with Cytotoxic and NO Production Inhibitory Activities from *Placolobium vietnamense*. *Molecules* **2022**, *27*, 4624. https://doi.org/10.3390/ molecules27144624

Academic Editor: Jacqueline Aparecida Takahashi

Received: 6 June 2022 Accepted: 12 July 2022 Published: 20 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Results and Discussion 2. Results and Discussion**  2.1. *Structural Elucidation of the Isolated Compounds*

#### *2.1. Structural Elucidation of the Isolated Compounds*

*Molecules* **2022**, *27*, x FOR PEER REVIEW 2 of 8

Chromatographic separation of benzil and isoflavone derivatives from *P. vietnamense* stems allowed for the isolation of eight compounds, including a new isoflavone derivative, placovinone A (**1**), and three new benzil derivatives, placovinones B-D (**2**–**4**), along with four known pyranoisoflavones (**5**–**8**) (Figure 1). The structures of all isolated compounds were elucidated based on NMR and HRMS spectral data, as well as a comprehensive comparison of their spectroscopic and physical data with values from the published literature. The known isolated pyranoisoflavones were characterized as ichthynone (**5**) [3], durmillone (**6**) [4], calopogoniumisoflavone B (**7**) [5], and 40 ,50 -dimethoxy-6,6 dimethylpyranoisoflavone (**8**) [6]. Chromatographic separation of benzil and isoflavone derivatives from *P. vietnamense*  stems allowed for the isolation of eight compounds, including a new pyranoisoflavone, placovinone A (**1**), and three new benzil derivatives, placovinones B‒D (**2**–**4**), together with four known pyranoisoflavones (**5**–**8**) (Figure 1). The structures of all isolated compounds were elucidated based on NMR and HRMS spectral data and a comprehensive comparison of their spectroscopic and physical data with values from the published literature. The known isolated pyranoisoflavones were characterized as ichthynone (**5**) [3], durmillone (**6**) [4], calopogoniumisoflavone B (**7**) [5], and 4′,5′‒ dimethoxy‒6,6‒dimethylpyranoisoflavone (**8**) [6].

**Figure 1.** Chemical structures of **1**‒**8**. **Figure 1.** Chemical structures of **1**–**8**.

Compound **1** was isolated as a colorless gum. The HRESIMS revealed a protonated molecular ion peak at *m/z* 367.1549 [M + H]+ (calcd for C22H23O5 367.1545) corresponding to the formula C22H22O5. The 1H NMR signal at *δ*H 8.42 (1H, s, H‒2) and 13C NMR signal at *δ*C 152.7 (C-2) were characteristic of the isoflavone skeleton [7]. The existence of AA′BB′ spin*-*system indicated *para-*substituted B*‒*ring. The presence of a 2,2‒dimethyldihydro pyran [8] and two methoxy substituents was identified from both 1H and 13C NMR spectra (Table 1). A singlet at *δ*H 7.32 (1H) was assigned to the aromatic proton H-5 on the basis of the long*-*range coupling to C*‒*4 (*δ*C 174.4), C*‒*7 (*δ*C 148.3), and C*‒*8a (*δ*C 109.9), observed in the HMBC spectrum (Figure 2). The methoxy group at *δ*H 3.84 (3H, s) was assigned as 6- OCH3 according to the HMBC correlation between 6*‒*OCH3 and C*‒*6 (*δ*C 147.1). The longrange correlations observed in the HMBC spectra of H-1′′ at *δ*H 2.87 (2H, t, *J* = 6.5 Hz) to C*‒*7 and C*‒*8a were key correlations that revealed the position of 2,2‒dimethylpyran moiety was fused to C‒7 and C‒8. Its 1D and 2D NMR spectral data were similar to those of 6*‒*methoxycalopogonlum isoflavone A [9], except for the replacement of a double bond at C*‒*1′′ and C*‒*2′′ of the 2,2‒dimethylpyran substituent in 6*‒*methoxycalopogonlum isoflavone A by a C*‒*C single bond in **1**. Based on the above spectral evidence, the structure of **1** was established and trivially named as placovinone A. Compound **1** was isolated as a colorless gum. The HRESIMS revealed a protonated molecular ion peak at *m/z* 367.1549 [M + H]<sup>+</sup> (calcd for C22H23O<sup>5</sup> 367.1545) corresponding to the formula C22H22O5. The <sup>1</sup>H NMR signal at *δ*<sup>H</sup> 8.42 (s, H-2) and <sup>13</sup>C NMR signal at *δ*<sup>C</sup> 152.7 (C-2) were characteristic of the isoflavone skeleton [7]. The existence of AA0BB0 spinsystem indicated *para*-substituted B-ring. The presence of a 2,2-dimethyldihydropyrano [8] and two methoxy substituents was identified from the <sup>1</sup>H and <sup>13</sup>C NMR spectral data (Table 1). A singlet resonance at *δ*<sup>H</sup> 7.32 was assigned to the aromatic proton H-5 on the basis of the long-range coupling to C-4 (*δ*<sup>C</sup> 174.4), C-7 (*δ*<sup>C</sup> 148.3), and C-8a (*δ*<sup>C</sup> 109.9), observed in the HMBC spectrum (Figure 2). The methoxy group *δ*<sup>H</sup> 3.84 (s) was assigned as 6-OCH<sup>3</sup> according to the HMBC correlation between 6-OCH<sup>3</sup> and C-6 (*δ*<sup>C</sup> 147.1). The longrange correlations observed in the HMBC spectrum of H-1" (*δ*<sup>H</sup> 2.87, t, *J* = 6.5 Hz) to C-7 and C-8a were key correlations that revealed the position of 2,2-dimethyldihydropyrano moiety was fused to C-7 and C-8, with the anticipated oxygenation at C-7 being supported by the HMBC correlation from H-5 to C-7. Its 1D and 2D NMR spectral data were similar to those of 6-methoxycalopogonlum isoflavone A [9], except for the replacement of a double bond at C-1" and C-2" of the 2,2-dimethylpyrano substituent in 6-methoxycalopogonlum isoflavone A by a C-C single bond in **1**. Based on the above spectral evidence, the structure of **1** was established and trivially named as placovinone A.


**Table 1.** <sup>1</sup>H (500 MHz) and <sup>13</sup>C (125 MHz) NMR spectroscopic data of **1** recorded in DMSO-*d*<sup>6</sup> (*δ* in ppm).

**Figure 2.** Key COSY (red bold line) and HMBC (blue arrow) correlations of **1**‒**4**. **Figure 2.** Key COSY (red bold line) and HMBC (blue arrow) correlations of **1**–**4**.

**Table 1.** 1H (500 MHz) and 13C (125 MHz) NMR spectroscopic data of **1** recorded in DMSO*‒d*6 (*δ* in ppm). **Position** *δ***H (***J* **in Hz)** *δ***<sup>C</sup> Position** *δ***H (***J* **in Hz)** *δ***<sup>C</sup>** 2 8.42, s 152.7 3′ 6.99, d (8.3) 113.6 3 122.8 4′ 158.9 4 174.4 5′ 6.99, d (8.3) 113.6 4a 115.9 6′ 7.52, d (8.3) 130.0 5 7.32, s 101.8 1′′ 2.87, t (6.5) 16.4 6 147.1 2′′ 1.87, t (6.5) 30.6 7 148.3 3′′ 75.9 8 109.9 4′′ 1.35, s 26.3 8a 149.5 5′′ 1.35, s 26.3 1′ 124.4 6*‒*OCH3 3.84, s 55.1 2′ 7.52, d (8.3) 130.0 4′*‒*OCH3 3.79, s 55.5 Compound **2** was obtained as a white amorphous powder. Its molecular formula was determined to be C23H24O8 based on a protonated molecular ion peak at *m/z* 429.1566 (calcd for C23H25O8 429.1549). The signal of a hydroxyl group at *δ*H 10.11 (1H, s, 2*‒*OH) in the 1H NMR spectrum, together with those of two carbonyl groups at *δ*C 190.7 (C*‒*7) and 191.4 (C*‒*8) in the 13C NMR spectrum, indicated that **2** was a derivative of 1,2*‒*diphenyl*‒* Compound **2** was obtained as a white amorphous powder. Its molecular formula was determined to be C23H24O<sup>8</sup> based on a protonated molecular ion peak at *m/z* 429.1566 (calcd for C23H25O<sup>8</sup> 429.1549). The signal of a hydroxyl group at *δ*<sup>H</sup> 10.11 (s, 2-OH) in the <sup>1</sup>H NMR spectrum, together with those of two carbonyl groups at *δ*<sup>C</sup> 190.7 (C-7) and 191.4 (C-8) in the <sup>13</sup>C NMR spectrum, indicated that **2** was a derivative of 1,2-diphenyl-1,2 ethanedione [10]. The <sup>1</sup>H and <sup>13</sup>C NMR spectral data (Table 2) further revealed the presence of a 2,2-dimethylpyrano fragment and four methoxy substituents. In the <sup>1</sup>H NMR spectrum, two singlet protons at *δ*<sup>H</sup> 6.76 and 7.41, were assigned to the two *para*-positioned aromatic protons H-3' and H-6' of the B-ring [11], indicating the B-ring of **2** with 20 ,40 ,50 -trimethoxy substituent. This was also supported by the strong correlations in the HMBC spectrum (Figure 2). The singlet of the aromatic proton at *δ*<sup>H</sup> 7.24 was identified as H-6 on the basis of the HMBC correlations from H-6 to C-1 (*δ*<sup>C</sup> 112.5), C-2 (*δ*<sup>C</sup> 149.7), C-4 (*δ*<sup>C</sup> 148.2), and C-7 (*δ*<sup>C</sup> 190.7). Consequently, the remaining methoxy group (*δ*<sup>H</sup> 3.84, s) was located at C-5, confirmed by the key HMBC correlation between 5-OCH<sup>3</sup> and C-5 (*δ*<sup>C</sup> 142.5). Hence the location of the 2,2-dimethylpyrano moiety was found to be at C-3 (*δ*<sup>C</sup> 108.9) and C-4, with the anticipated oxygenation at C-4 being confirmed by the HMBC correlation from H-6 to C-4. A careful comparison of the <sup>1</sup>H and <sup>13</sup>C NMR spectral data (Table 2) of **2** with dielsianone [12] identified similar signals, distinguished by the presence of two methoxy groups at C-20 and C-50 . The existence of these two methoxy substituents was confirmed by the HMBC correlations from 20 -OCH<sup>3</sup> (*δ*<sup>H</sup> 3.33, s) and 5<sup>0</sup> -OCH<sup>3</sup> (*δ*<sup>H</sup> 3.89, s) to C-2<sup>0</sup> (*δ*<sup>C</sup> 156.8) and C-50 (*δ*<sup>C</sup> 155.6), respectively (Figure 2). From the aforementioned results, the structure of **2** was identified and named as placovinone B.

1,2*‒*ethanedione [10]. The 1H and 13C NMR spectral data (Table 2) further revealed the presence of a 2,2*‒*dimethylpyran fragment and four methoxy substituents. In the 1H NMR

2′,4′,5′*‒*trimethoxy substituent. This was also supported by the strong correlations in the HMBC spectrum (Figure 2). The singlet of the aromatic proton at *δ*H 7.24 was identified as H*‒*6 on the basis of the HMBC correlations from H*‒*6 to C*‒*1 (*δ*C 112.5), C*‒*2 (*δ*C 149.7), C*‒*4 (*δ*C 148.2), and C*‒*7 (*δ*C 190.7). Consequently, the remaining methoxy group (*δ*H 3.84, s) was located at C*‒*5, confirmed by the key HMBC correlation between 5*‒*OCH3 and C*‒*5 (*δ*<sup>C</sup> 142.5). Hence the location of the 2,2*‒*dimethylpyran moiety was found to be at C*‒*3 (*δ*<sup>C</sup> 108.9) and C*‒*4, with the anticipated oxygenation at C*‒*4 being confirmed by the HMBC correlation from H*‒*6 to C*‒*4. A careful comparison of the 1H and 13C NMR spectral data (Table 2) of **2** with dielsianone [12] identified similar signals, distinguished by the pres-


**Table 2.** <sup>1</sup>H (600 MHz) and <sup>13</sup>C (125 MHz) NMR spectroscopic data of **2**–**4** recorded in DMSO-*d*<sup>6</sup> (*δ* in ppm).

Compound **3** was isolated as a white amorphous powder. Its molecular formula, C23H26O7, was determined from its protonated molecular ion peak at *m/z* 415.1759 [M + H]<sup>+</sup> (calcd for C23H27O<sup>7</sup> 415.1757). This was further confirmed by the <sup>13</sup>C NMR spectral data, which disclosed one methylene, two methyl, two olefinic, three aromatic methine, four methoxy, and ten quaternary carbons. The spectroscopic <sup>1</sup>H and <sup>13</sup>C NMR patterns of **3** (Table 2) were very similar to those of **2**, with the only difference being that the keto carbonyl group at C-8 in **2** (*δ*<sup>C</sup> 191.4) was replaced by a methylene substituent in **3**. This deduction was supported by the HMBC correlations from H-8 (*δ*<sup>H</sup> 4.20, s) to C-7 (*δ*<sup>C</sup> 202.9) and C-10 (*δ*<sup>C</sup> 114.2). Based on the above spectral evidence, compound **3** was identified and named placovinone C.

Compound **4** was obtained as a white amorphous powder. The molecular formula C21H22O<sup>5</sup> was obtained from its HRESIMS, which showed a protonated molecular ion peak at *m/z* 355.1553 [M + H]<sup>+</sup> (calcd for C21H23O<sup>5</sup> 355.1545). <sup>13</sup>C NMR and HSQC spectra of **4** indicated 21 signals, including one carbonyl, one methylene, two methyl, two methoxy, seven methine, and eight quaternary carbons. Two signals at *δ*<sup>H</sup> 7.21 (d, *J* = 8.7 Hz, H-20 , 60 ) and 6.88 (d, *J* = 8.7 Hz, H-30 , 50 ) appearing as an AA0BB0 type confirmed the presence of a simple *para*-substituted B-ring, with a methoxy group (*δ*<sup>H</sup> 3.47, s) being positioned at C-40 (*δ*<sup>C</sup> 158.2). The careful comparison of the <sup>1</sup>H and <sup>13</sup>C NMR spectral data (Table 2) of **4** was shown to be similar to those of **3**, differing only in the absence of two methoxy groups at C-20 (*δ*<sup>C</sup> 130.7) and C-5<sup>0</sup> (*δ*<sup>C</sup> 114.1) on the B-ring of **4**, which was supported by the COSY and HMBC correlations (Figure 2). On the basis of these spectral data, the structure of **4** was unambiguously established and named as placovinone D.

#### *2.2. Cytotoxicity*

The cytotoxicity of each isolated compound against Hep G2 cell line was assessed [13–15] and the IC<sup>50</sup> values are listed in Table 3. Compounds **1**–**8** exhibited different degrees of cytotoxicity toward Hep G2 cell line. Among them, compound **1** exhibited the most significant cytotoxicity against Hep G2 cell line with an IC<sup>50</sup> value of 8.0 µM. Compounds **2**–**4** and **8** showed moderate cytotoxicity with the IC<sup>50</sup> values of 19.8, 22.9, 23.4, and 35.6 µM, respectively, while compounds **5**–**7** exhibited weak cytotoxicity with the IC<sup>50</sup> values of 99.1, 71.6, and 66.6 µM, respectively. Based on the above cytotoxic results, the presence of the 2,2-dimethyldihydropyrano ring in the case of **1** might be responsible for enhancing the activity.

**Table 3.** Cytotoxicity against Hep G2 cells and inhibition of NO production in macrophage RAW 264.7 cells of **1**–**8**.


a IC<sup>50</sup> values were expressed as the mean values of three experiments <sup>±</sup> SD. <sup>b</sup> Positive control.

#### *2.3. Inhibition of Nitric Oxide Production*

To determine the inhibitory effects of the isolated compounds on NO production (Table 3), LPS-stimulated RAW 264.7 cells were treated with various concentrations of tested compounds [16]. Additionally, the viability of RAW 264.7 cells using an MTT assay to avoid the cytotoxic effects of the isolated compounds was evaluated. Among eight isolated compounds, compounds **1** and **4** highly inhibited NO production in RAW 264.7 cells with the IC<sup>50</sup> values of 13.7 and 15.5 µM, respectively, whereas compounds **2**, **3**, and **8** moderately inhibited NO production with the IC<sup>50</sup> values of 31.0, 47.4, and 54.7 µM, respectively. Compounds **1** and **4** demonstrated cytotoxicity toward RAW 264.7 cells with the IC<sup>50</sup> values of 79.2 and 42.6 µM, respectively, while most of the other compounds showed no obvious cytotoxicity (IC<sup>50</sup> >100 µM). These results demonstrate that the presence of the *para*-substituted B-ring of **1** and **4** might be responsible for inhibiting NO production.

#### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

The NMR spectra were recorded on Bruker AvanceNEO 600 MHz and Bruker Avance III™ HD 500 MHz NMR spectrometers in DMSO-*d*<sup>6</sup> (Merck, Darmstadt, Germany). Optical rotations were measured on a A.KRÜSS Optronic P8000 polarimeter (KRÜSS, Hamburg, Germany). The IR data were obtained with a Jasco 6600 FT-IR spectrometer using an ATR technique (Jasco, Japan). The HRESIMS spectral data were generated with a X500<sup>R</sup> QTOF model mass spectrometer (Sciex, Framingham, MA, USA) and Dionex Ultimate 3000 HPLC system hyphenated with a QExactive Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA). Silica gel 70–230 mesh (Merck) and Sephadex LH-20 gel (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography.

#### *3.2. Plant Material*

The stems of *P. vietnamense* were collected in Dak Nong province, Vietnam, in February 2017. The plant material was identified by botanist Vo Van Chi (former lecturer at the University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam). A voucher specimen (No. SGU-A001) has been deposited in the Herbarium of the Laboratory of Chemistry-Biology-Environment, Sai Gon University, Ho Chi Minh City, Vietnam.

#### *3.3. Extraction and Isolation*

The air-dried *P. vietnamense* stems (23 kg) were powdered prior to being extracted with 95% EtOH (45 L × 5) at room temperature. The filtered solution was concentrated in vacuo to afford EtOH crude extract (1200 g). This crude extract was suspended in water and partitioned with *n*-hexane and then EtOAc to yield *n*-hexane (271.2 g) and EtOAc (301.3 g) extracts, respectively. The *n*-hexane extract was subjected to silica gel column chromatography (CC) and eluted with *n*-hexane–EtOAc (9:1–0:10, *v/v*) and then EtOAc– MeOH (10:0–0:10, *v/v*). Based on their TLC behavior, the eluted fractions were grouped into fractions HEX.1–HEX.7. Fraction HEX.4 (34.5 g) was subjected to further silica gel CC and eluted with *n*-hexane–EtOAc (8:2, *v/v*) to give subfractions HEX.4.1–HEX.4.8. Subfraction HEX.4.1 (3.0 g) was subjected to silica gel CC and eluted with *n*-hexane–EtOAc (85:15, *v/v*) to yield **3** (7.0 mg), **5** (8.0 mg), and **6** (9.7 mg). Subfraction HEX.4.2 (0.9 g) was further purified using silica gel CC and eluted with *n*-hexane–EtOAc (8:2, *v/v*) to yield **2** (6.5 mg), **7** (6.4 mg), and **8** (11.4 mg). Subfraction HEX.4.3 (1.1 g) was selected for further purification using Sephadex LH-20 gel CC and eluted with MeOH to afford **1** (5.8 mg) and **4** (6.4 mg).

Placovinone A (**1**). Colorless gum. UV (CH3OH) *λ*max (log *ε*) 210 (4.49), 231 (4.25), 278 (4.81), 334 (3.47) nm; IR (ATR) *ν*max 2975, 1718, 1619, 1457, 1343, 1279, 1203, 1150, 1013, 757 cm−<sup>1</sup> ; HRESIMS *m/z* 367.1549 [M + H]<sup>+</sup> (calcd for C22H23O<sup>5</sup> 367.1545); <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz) and <sup>13</sup>C NMR (DMSO-*d*6, 125 MHz) see Table 1.

Placovinone B (**2**). White amorphous powder. UV (CH3OH) *λ*max (log *ε*) 250 (4.39), 270 (4.72), 296 (4.30), 337 (3.18) nm; IR (ATR) *ν*max 3392, 2977, 2904, 1713, 1635, 1451, 1372, 1288, 1246, 900 cm-1; HRESIMS *m/z* 429.1566 [M + H]<sup>+</sup> (calcd for C23H25O<sup>8</sup> 429.1549); <sup>1</sup>H NMR (DMSO-*d*6, 600 MHz) and <sup>13</sup>C NMR (DMSO-*d*6, 125 MHz) see Table 2.

Placovinone C (**3**). White amorphous powder. UV (CH3OH) λmax (log ε) 205 (4.07), 272 (4.87), 339 (2.98) nm; IR (ATR) νmax 3394, 2977, 2889, 1710, 1642, 1447, 1333, 1289, 1216, 763 cm−<sup>1</sup> ; HRESIMS *m/z* 415.1759 [M + H]<sup>+</sup> (calcd for C23H27O<sup>7</sup> 415.1757); <sup>1</sup>H NMR (DMSO-*d*6, 600 MHz) and <sup>13</sup>C NMR (DMSO-*d*6, 125 MHz) see Table 2.

Placovinone D (**4**). White amorphous powder. UV (CH3OH) λmax (log *ε*) 205 (4.11), 270 (4.87), 333 (3.06) nm; IR (ATR) *ν*max 3395, 2977, 2896, 1712, 1643, 1448, 1339, 1287, 1218, 763 cm−<sup>1</sup> ; HRESIMS *m/z* 355.1553 [M + H]<sup>+</sup> (calcd for C21H23O<sup>5</sup> 355.1545); <sup>1</sup>H NMR (DMSO-*d*6, 600 MHz) and <sup>13</sup>C NMR (DMSO-*d*6, 125 MHz) see Table 2.

#### *3.4. Cytotoxicity Assay*

According to a previous procedure [17], the cytotoxic evaluation of **1**–**8** against the growth of human hepatocellular carcinoma (Hep G2) cell line was carried out. The positive control was ellipticine, a powerful anticancer medication with various modes of action. The cancer cells were grown in Dulbecco's Modified Essential Medium (DMEM) at 37 ◦C in a 5 % CO<sup>2</sup> environment with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, and 1% L-glutamine. The investigated compounds were added at concentrations ranging from 0.5 to 128 µg/mL by dissolving in DMSO (20 mg/mL), and the incubation was carried out once more for 72 h under the same conditions. Following the procedure, an MTT solution (10 µL, 5 mg/mL) was added to each well. The percentage of cell viability vs. sample concentration was plotted using SigmaPlot 10 (Systat Software Inc., San Jose, CA, USA) to calculate the IC<sup>50</sup> values.

## *3.5. Inhibition of Nitric Oxide Production Assay* 3.5.1. Cell Culture

RAW 264.7 cells were stocked in Dulbecco's Modified Essential and grown at the condition of 37 ◦C in DMEM supplemented with 10% heat-inactivated FBS, streptomycin sulfate (100 µg/mL), and penicillin (100 units/mL) in a humidified environment of 5% CO2. The RAW 264.7 cells were pre-incubated every two days.

#### 3.5.2. Cell Viability Assay on RAW 264.7 Cells

The cell viability assay was used to determine the cytotoxic effect of the isolated compounds on RAW 264.7 cells. At a density of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well, RAW 264.7 cells were seeded on a 96-well plate and allowed to adhere for 4 h. Then, the cells were treated with 0.5% DMSO, celastrol, and isolated compounds at the indicated concentrations. Celastrol was used as a positive control [16]. After incubating 24 h, the viable cells were measured with a colorimetric assay based on the mitochondria's ability in viable cells to reduce MTT [18]. The viability cells were treated with vehicle only and were defined as 100% viable. [OD<sup>570</sup> (treated cell culture) × 100]/OD<sup>570</sup> was the formula used to determine the percentage of macrophage surviving cells after treatment (vehicle control).

#### 3.5.3. Measurement of Nitric Oxide (NO) Production

The RAW 264.7 cells were stimulated with or without 1 µg/mL of LPS (lipopolysaccharide), which was purchased from Sigma Chemical Co. (St. Louis, MO, USA), for 24 h with or without 0.5% DMSO, celastrol, and isolated compounds at the indicated concentrations. The culture supernatant (100 µL) was then reacted with 100 µL of Griess reagent [16]. After the Griess assay, the remaining cells were used to screen for their viability using colorimetric assay-MTT (Sigma Chemical Co., St. Louis, MO, USA).

#### **4. Conclusions**

In conclusion, we have conducted the successful isolation of eight compounds, including a new isoflavone derivative (**1**) and three new benzil derivatives (**2**–**4**), together with four known pyranoisoflavones (**5**–**8**) from *P. vietnamense* stems. To the best of our knowledge, compounds **1**–**8** were isolated for the first time from the genus *Placolobium*. The biological evaluations showed that **1** exhibited the most significant cytotoxicity toward Hep G2 cell line and the strongest inhibitory activity against the LPS-induced NO production. According to these investigation results, the structure of **1** is a promising candidate and could be used as a template for discovering potential anticancer and anti-inflammatory agents.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27144624/s1, Figures S1–S23: HRESIMS, 1D, and 2D NMR spectra of **1**–**4**.

**Author Contributions:** Conceptualization, J.S. and L.T.M.D.; methodology, L.T.M.D. and T.T.N.H.; formal analysis, J.S.; data curation, J.S. and L.T.M.D.; writing—original draft preparation, J.S. and L.T.M.D.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Thammasat University Research Unit in Natural Products Chemistry and Bioactivities.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The supporting information can be found in the Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


## *Article* **Two Natural Flavonoid Substituted Polysaccharides from** *Tamarix chinensis***: Structural Characterization and Anticomplement Activities**

**Yukun Jiao, Yiting Yang, Lishuang Zhou, Daofeng Chen \* and Yan Lu \***

School of Pharmacy, Institutes of Integrative Medicine, Fudan University, Shanghai 201203, China; 17111030034@fudan.edu.cn (Y.J.); 17211030024@fudan.edu.cn (Y.Y.); 20111030064@fudan.edu.cn (L.Z.) **\*** Correspondence: dfchen@shmu.edu.cn (D.C.); luyan@fudan.edu.cn (Y.L.)

**Abstract:** Two novel natural flavonoid substituted polysaccharides (MBAP-1 and MBAP-2) were obtained from *Tamarix chinensis* Lour. and characterized by HPGPC, methylation, ultra-high-performance liquid chromatography-ion trap tandem mass spectrometry (UPLC-IT-MS<sup>n</sup> ), and NMR analysis. The results showed that MBAP-1 was a homogenous heteropolysaccharide with a backbone of 4)-β-D-Glc*p*-(1→ and →3,4,6)-β-D-Glc*p*-(1→. MBAP-2 was also a homogenous polysaccharide which possessed a backbone of →3)-α-D-Glc*p*-(1→, →4)-β-D-Glc*p*-(1→ and →3,4)-β-D-Glc*p*-2-OMe-(1→. Both the two polysaccharides were substituted by quercetin and exhibited anticomplement activities in vitro. However, MBAP-1 (CH50: 0.075 ± 0.004 mg/mL) was more potent than MBAP-2 (CH50: 0.249 ± 0.006 mg/mL) and its reduced product, MBAP-1R (CH50: 0.207 ± 0.008 mg/mL), indicating that multiple monosaccharides and uronic acids might contribute to the anticomplement activity of the flavonoid substituted polysaccharides of *T. chinensis*. Furthermore, the antioxidant activity of MBAP-1 was also more potent than that of MBAP-2. In conclusion, these two flavonoid substituted polysaccharides from *T. chinensis* were found to be potential oxidant and complement inhibitors.

**Keywords:** *Tamarix chinensis* Lour.; flavonoid substituted polysaccharides; structural characterization; anticomplement activity; quercetin

## **1. Introduction**

The complement system, as the first defense line of human immune system, plays an irreplaceable role in numerous diseases [1]. Our previous study showed that the overactivation of complement system is involved with H1N1 induced pneumonia in mice [2]. Moreover, the significant elevation of peripheral complement has been recognized as a hallmark of respiratory distress syndrome associated with severe sepsis, cytokine storm, and multiple organ failure [3]. The autopsy results of COVID-19 patients also suggested that hyper complementation in plasma resulted in alveolar capillary wall damage with increased vascular permeability and further enhanced release of inflammatory mediators, and then intensified tissue damage [4]. In addition, two complement inhibitors, eculizumab and compstatin, have been suggested as potential treatments for COVID-19 [5]. In our previous research, anticomplement polysaccharides and flavonoids from several medicinal plants could significantly alleviate lung injury and increase the survival rate of H1N1 induced mice, and were non-toxic [2,6,7]. Hence, the medicinal plants provided a new resource of complement inhibitors for the treatment of viral pneumonia.

*Tamarix chinensis* Lour. (Tamaricaceae) has been widely used to treat rheumatoid arthritis, measles (Chinese Pharmacopoeia, 2020), and measles complicated with pneumonia [8]. Its flavonoids, triterpenoids, organic acids, and volatile oils showed anti-inflammatory, bacteriostatic, antioxidant, and hepatoprotective effects [9]. However, there were no reports about *T. chinensis* polysaccharides. In our preliminary studies, the crude polysaccharides of *T. chinensis*, MBAP90, showed significant anticomplement activity with CH<sup>50</sup> value of

**Citation:** Jiao, Y.; Yang, Y.; Zhou, L.; Chen, D.; Lu, Y. Two Natural Flavonoid Substituted Polysaccharides from *Tamarix chinensis*: Structural Characterization and Anticomplement Activities. *Molecules* **2022**, *27*, 4532. https:// doi.org/10.3390/molecules27144532

Academic Editor: Jacqueline Aparecida Takahashi

Received: 2 July 2022 Accepted: 12 July 2022 Published: 15 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

0.186 ± 0.003 mg/mL. Interestingly, MBAP90 exhibited the characteristic color reaction of flavonoids even after deproteinization and dialysis (cutting off MW: 5000 Da). The <sup>1</sup>H-NMR signals at δ 6.5–8.0 also indicated that MBAP90 might contain flavonoid substituted polysaccharides.

In recent years, numerous methods have been applied to graft flavonoids and polysaccharides [10]. Some synthesized flavonoid-polysaccharide conjugates possessed unique advantages compared with flavonoids or polysaccharides. For example, quercetin-grafted carboxymethyl chitosan was amphiphilic with a low critical micelle concentration and good stability [11]. Quercetin-grafted hyaluronic was designed as tumor cell-targeted prodrug for its significant intestinal permeability, oral bioactivity and antitumor efficacy [12]. However, there were no reports of natural flavonoid substituted polysaccharides.

To explore anticomplement polysaccharides in *T. chininsis* and their anticomplement activities, MBAP90 was further purified by DEAE-cellulose and Sepharyl S-200, which led to the isolation of two novel homogenous polysaccharides, MBAP-1 and MBAP-2. This paper describes their structural characterization and anticomplement activities. As oxidative stress is vital for inflammatory responses in viral pneumonia, antioxidant activities were also determined herein as well [13].

#### **2. Results**

#### *2.1. Isolation and Purification of MBAP-1 and MBAP-2*

MBAP-1 and MBAP-2 were isolated from the most potent anticomplement fraction (Fr. 2) of MBAP90, and the yields were 0.14% and 0.61%, respectively. The detailed elution curves are shown in Figure 1A.

**Figure 1.** The eluted profiles and HPSEC-MALLS-RI results. (**A**) The eluted profile of MBAP90 on DEAE-52 column and Fr. 2 on Sepharyl S-200 column. (**B**) Superimposed spectra detected using RI and LC at angle of 90◦ on HPSEC-MALLS-RI. (**C**) Molar mass distribution detected by HPSEC-MALLS-RI.

#### *2.2. Homogeneity and Molecular Weight Assessment*

The homogeneity was evaluated by HPGPC-ELSD. As shown in Figure S1 (Supplementary Materials), MBAP-1 and MBAP-2 both showed one single narrow peak, indicating that they were both homogenous. As displayed in Figure 1B,C, the two polysaccharides were both further confirmed to be homogenous using HPSEC-MALLS-RI. Moreover, the results suggested that the relative molecular weights of MBAP-1 and MBAP-2 were 269.3 and 46.5 kDa, respectively. The detailed parameters of MBAP-1 and MBAP-2 were summarized in Table 1.


**Table 1.** The molecular parameters of MBAP-1 and MBAP-2 determined by SEC-MALLS-RI.

#### *2.3. Monosaccharide Composition and Absolute Configuration Analysis*

The monosaccharide composition results of MBAP-1 and MBAP-2 are presented in Figure 2A. Obviously, MBAP-1 was composed of glucose, galactose, arabinose, glucuronic acid, and galacturonic acid with the molar ratio of 54.54:4.21:18.18:4.87:4.21. MBAP-2 was mainly consisted of glucose. However, an unknown peak was presented at 24.67 min, which was further identified by GC-MS. The unknown monosaccharide was attributed as 2-*O*-methyl glucose by ion fragments of its alditol acetate (Figure S2). Thus, MBAP-2 was mainly composed of glucose and 2-*O*-methyl glucose with a molar ratio of 88.41:11.59. The *w*/*w* (%) ratio of each monosaccharide in two polysaccharides is presented in Table 2.

**Figure 2.** The chromatograms of monosaccharide composition (**A**), monosaccharide absolute configuration analysis (**B**) and UPLC-MS identification results of substituted flavonoids (**C**,**D**) of MBAP-1 and MBAP-2.


**Table 2.** The primary chemical characteristics of MBAP-1 and MBAP-2.
