Raman Spectroscopy: A Key Analytical Tool for New Drugs Research and Development

Chapter 6

Raman Spectroscopy: A Key Analytical Tool for New Drugs Research and Development Simona Cıˆnta˘ Pıˆnzaru*,1, A. Fa˘la˘maș† and C.A. Dehelean‡ *

Biomolecular Physics Department, Babeș-Bolyai University, Cluj-Napoca, Romania National Institute for R&D of Isotopic and Molecular Technologies, Cluj-Napoca, Romania ‡ Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timișoara, Romania 1 Corresponding author:e-mail: [email protected] †

Chapter Outline Introduction Infrared and Raman Spectroscopy: Basic Considerations Raman Spectroscopy Identification the Main Triterpenes of the Lupane Group in Birch Bark (Betula pendula Roth) From Romanian Flora Birch Bark Extraction Products: Raman Spectroscopy Analysis

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Pharmaceutical Formulation of Betulin and Birch Bark Extracts Evaluation of Betulin’s Therapeutic Effect on Mice Skin by Means of Raman Spectroscopy Concluding Remarks References

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INTRODUCTION Natural products have always been a major source of new drugs and have been used for treating various diseases for centuries. Even though they have seen a temporary regress due to organic synthesis chemistry, and the development of synthetic derivatives which have occupied a significant place in the area of drug discovery, the risks posed by synthetic products, their side effects, and even lack of therapeutic response, have led to and increased and renewed interest in the discovery of natural therapies. Birch species of the genus Betula, representing trees abundant in the temperate climates of the northern hemisphere and comprising numerous taxa [1,2], are visually recognized by the appearance of their outer, white bark, which exfoliates circularly, being structured from cells with large intercellular Studies in Natural Products Chemistry, Vol. 61. https://doi.org/10.1016/B978-0-444-64183-0.00006-3 Copyright © 2018 Elsevier B.V. All rights reserved.

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spaces. The outer bark of the birch tree has been extensively used since ancient times as a healing agent or for producing household utensils, or artistic pieces [3]. In the past decades, birch bark has been the subject of consistent scientific research [4,5] in various fields including the pharmaceutical and cosmetics industries. The outer bark is rich in pentacyclic triterpenes with lupane skeletons, such as betulin, betulinic acid, lupeol, and ursolic and oleanolic acids. The buds of the tree contain vegetal hormones, mineral salts, triterpenes, and essential oils; the leaves contain saponins [6]. The vast volume of biomedical research on pentacyclic triterpenes began with the significant contributions of Pisha et al. [7] and Steele et al. [8]—they showed the anticancer and antimalarial properties of betulinic acid. Since then, many publications have followed, highlighting the apoptosis activity of pentacyclic triterpenes with lupane skeletons on melanoma cells [9,10], neuroblastomas [11], and brain tumor cells [12], as well as their activity against HIV [13] and their antiinflammatory activity [14]. These publications led to the inclusion of betulinic acid in phase II clinical trials for the treatment of melanoma [7]. Betulin (lup-20(29)-ene-3,28-diol) is a natural compound which can be easily extracted from birch bark using various methods [15–19]. Despite the fact that betulin has not shown comparable effectiveness in terms of its anticancer activity to betulinic acid [20], a proapoptotic and an inhibitory effect was observed in studies of lung cancer cells [21]; important antitumor activity was reported when used in association with cholesterol [22]; and its antibacterial, antifungal, and antiviral effects have been reported [23]. Moreover, its pharmaceutical properties are the subject of ongoing research [4,6,24]. A major hindrance for studying the biological activity of betulin and betulinic acid, and formulating pharmaceutical medications, is their low water solubility. Cao et al. investigated the solubility of betulin in various organic solvents at different temperatures and offered a guide which could be used during the processes of crystallization by industry [25]. Other reported methods to increase its solubility and/or bioavailability rely on nanoemulsion formulations [26], cyclodextrin guest–host-type inclusions [27–29], an oil suspension intraperitoneal delivery route using sesame oil in rats or subcutaneous delivery in dogs [30], or modifications of their molecular structures using medicinal chemistry approaches, without loss of their biological properties [31,32]. Given the promising biological activities of triterpenes from birch bark, their extensive and intensive research, and efforts to translate Raman spectroscopybased tools and techniques to biomedical and pharmaceutical sectors, we summarize in this chapter several applications regarding Raman spectroscopy techniques which can be used for: (1) the identification of triterpenes of the lupane group in natural resources; (2) the characterization of extraction products; (3) their pharmaceutical formulations; and (4) the evaluation of their therapeutic effects as anticancer agents. Fig. 6.1 shows the steps already accomplished by means of Raman spectroscopy techniques for new

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FIG. 6.1 Graphical sketch showing the steps which can be accomplished by means of Raman spectroscopy techniques for new betulin-based drug source identification, extraction product evaluation, pharmaceutical formulation, and testing on animal models. Fourier transform (FT)-Raman, confocal Raman microspectroscopy (CRM), surface-enhanced Raman scattering (SERS), and depth profiling by CRM have been employed. Adapted from Ref. [33].

betulin-based drug source identification, extraction product evaluation, pharmaceutical formulation, and testing on animal models. New drug discoveries originating with plants is a complex process involving several steps such as plant identification, collection, extraction, isolation, and elucidation of the chemical structure of its active components. All this needs to be followed by the production of various formulations, delivery techniques, research into therapeutic activities, and toxicity studies.

INFRARED AND RAMAN SPECTROSCOPY: BASIC CONSIDERATIONS Vibrational spectroscopy, comprising two basic analytical techniques, infrared (IR) and Raman spectroscopy, is widely used as a nondestructive tool to obtain valuable information on the molecular composition and dynamics of materials, from simple chemical species to complex biological samples like cells or tissue, including raw natural products. Both infrared and Raman spectroscopy are expanded in multiple, derived techniques, adapted to or suitable for various macrosample or microsample characteristics and properties. Infrared spectroscopy measures the absorbed infrared radiation by molecules, and depending on the radiation frequency range is divided into nearinfrared (NIR), mid-infrared (MIR), and far-infrared (FIR) spectroscopy. Raman spectroscopy is based on the inelastic light scattering of materials. The inelastically scattered light, known as Raman spectrum, comprises

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characteristic vibrational modes of molecules which are usually excited by laser radiation. Energy transitions involving changes in vibrational states of molecules are called vibrational transitions and are described by vibrational Raman spectroscopy and IR absorption. The frequency of vibrational movements is comprised in the infrared range of the electromagnetic spectrum. Other transitions involving changes in rotational states only (gases in most cases) refer to rotational spectroscopy, while those comprising both vibrational and rotational states refer to rotational–vibrational spectroscopy. Absorption or emission of radiation (transition between electronic states) may sometimes occur alongside vibrational changes. These cases are described by vibronic (vibrational + electronic) transitions. Vibronic transitions generate more complex spectra and their explanation is governed by typical selection rules and the Frank–Condon principle, based on quantum mechanics theory. For a detailed and systematic description of infrared and Raman spectra, newcomers to the field of optical spectroscopy are strongly encouraged to consult the most comprehensive spectroscopy books published [33–35]. Raman and IR spectroscopy are complementary techniques, probing the same range of energies with different selection rules. While the absorption of IR radiation is conditioned by the existence of a dipole moment associated with a molecular vibration, Raman scattering depends on the modification of a molecule’s polarizability (induced dipoles as a response of bound atoms in molecules to an external electromagnetic field) and subsequent scattering of photons. The resulting spectra offer a unique chemical fingerprint for the investigated sample and these vibrational methods are able to detect sensitive modifications at molecular levels. Due to more recent technological developments, i.e., high-performance, cost-effective lasers; notch filters to suppress Rayleigh scattering, high-resolution dispersive holographic gratings; sensitive detectors based on charge-coupled devices; fiber-optic sampling; and integrated, flexible Raman spectrometers which can be combined with microscopes in micro-Raman instruments [36], Raman spectroscopy has transitioned to a widely used analytical technique in broad and multidisciplinary fields derived from physical, chemical, and life sciences. Raman spectroscopy is capable of detecting subtle biochemical changes in cells or tissues induced by diseases or drug treatments—these biochemical changes generate significant changes in Raman signals [37]. The application of Raman spectroscopy in the pharmaceutical field has shown immense potential for the identification and quantification of active drugs and excipients in pharmaceutical formulations [38,39]. By considering more of its advantages, such as minimal or lack of sample preparation and rapid, nondestructive investigation (no labels required), the technique has gained a lot of interest in the pharmaceutical and biomedical fields, advancing it to the point where its diagnostic accuracy and speed are compatible with its clinical use [40].

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RAMAN SPECTROSCOPY IDENTIFICATION OF THE MAIN TRITERPENES OF THE LUPANE GROUP IN BIRCH BARK (Betula pendula ROTH) FROM ROMANIAN FLORA Although abundant in birch trees, the potential of Romanian flora as an important resource of triterpenes has been completely ignored. The increased demand for triterpene products is related to biomedical research, the pharmaceutical industry, cosmetics, and nutraceuticals. Although rather expensive, triterpenes of the lupane group are increasingly involved in nanomedicine and targeted medicinal research, particularly anticancer research [4,6,24]. Therefore we summarized here the expanded Raman spectroscopy applications for the evaluation of pentacyclic triterpene local resources from two birch forestry areas in Romania. As birch is abundantly spread within spontaneous Romanian flora, and due to its important resources not only related to the wood industry, but for pharmaceutical, cosmetic, and natural health care products, a method for predicting the highest extraction yields in a nondestructive, rapid, and sensitive manner is greatly needed. Moreover, pharmaceutical, cosmetic, health care, or nutraceutical stakeholders are continuously seeking fast, effective, and reliable methods for the inexpensive validation of new products. Since raw bark content will determine the quality of the final extract, it is extremely important to be able to identify triterpene-rich bark species as well as suggesting effective extraction protocols. So far, there is scant information regarding the possible correlation of triterpene content in birch bark with geoclimatic factors, tree birch species, age, or environmental conditions. Holonec et al. evaluated the betulin and betulinic acid content in birch bark collected from 10 distinct points divided between two areas, meadow and forest (specific premountain or mountain vegetation containing pine, larch, spruce fir, and other mixed species) in the Western Carpathians, using HPLC with UV detection [41]. The authors identified a higher content of betulin compared to betulinic acid, but with significant differences among the investigated areas. The mean concentration of betulin relative to bark was 126.85
12.56 mg/g in the samples from the first area and 89.84
8.43 mg/g in the second. However, it was reported that the percentage ratio of betulinic acid to betulin of 11.23% was higher in the second harvested area. Although the number of samples was limited for robust statistical analysis purposes, the findings suggested significant differences in birch bark content between biologically similar birch trees from distinct geoclimatic conditions. O’Connell et al. identified betulin and lupeol in the outer bark of four different Betula species and found betulin levels ranging from 5.0% to 22.0% [42]. Guo et al. [43] reported on a larger scale study, by investigating 48 sites of pure birch forestry from northeast China and harvesting exfoliated bark from 20 to 30 adult trees (Betula platyphylla Suk.) per site. Betulin and lupeol

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content was found to be higher in areas with increased temperatures and precipitation, and lower in areas with increased relative humidity. The authors concluded that the average betulin concentration found in their study was 80% higher than found in the birch trees from the Western Carpathians [43]. However, comparative results should take into account other variables like extraction protocol efficiency, sampling and treatment, dryness status, etc. Moreover, they concluded that a 113% higher content was obtained compared to contents from other studies in northeast China (113–202 mg/g betulin content reported by Zhao et al. [44]). The study [43] failed to establish correlations between the triterpene content and geographical gradients. Using FT-Raman and FT-IR spectroscopy in conjunction with gas chromatography–mass spectrometry (GC-MS) Cinta Pinzaru et al. [45] characterized and differentiated for the first time raw, exfoliated bark and the corresponding natural extract products obtained from B. pendula Roth species, abundant in Romania. The aim of this investigation was to evaluate the potential of FT-vibrational spectroscopy for comparative estimation of the main pentacyclic triterpene content in raw birch bark and in its corresponding final extracts. Exfoliated outer bark samples were harvested from mature trees populating the forested area in the Aninei Mountains in the Banat region of Romania (Fig. 6.2). Voucher samples were deposited in the herbarium of the Department of Pharmaceutical Botany of the Faculty of Pharmacy, University of Medicine and Pharmacy, Timisoara, Romania. An additional study focused on birch bark originating from a different geographical area in the Bucovina region of Romania (Fig. 6.2). The locations selected for bark harvesting were either natural reservations or protected parks—untouched by deforestation or pollution. B. pendula Roth, also known as silver birch or European white birch, was selected for the investigation. The spectral response of bark from different birch tree specimens was gathered in a spectral database. Fig. 6.3 presents an example of a data set consisting of the FT-Raman spectra of raw bark samples collected from various birch trees in the Apuseni Mountains in Romania, while Fig. 6.4 shows the FT-Raman spectra of birch bark harvested from three sites encoded DR (Runc Hill), DN (Black Hill), and VD (Vatra Dornei Park) from the Bucovina area of northern Romania. The spectra of the bark samples presented a fluorescence background, probably due to fluorescent components and other impurities in the bark. The spectra displayed in Fig. 6.4 are baseline corrected. The main Raman bands observed in the spectra are located at 2925, 2854, 1633, 1601, 1440, and 1196 cm1. When compared with the reference FT-Raman spectrum of betulin (Fig. 6.4), one of the main triterpenes found in birch bark, the spectra of the bark shows many similarities, such as the broad band at 1633 cm1 comprising the 1642 cm1 shoulder from betulin, the 1440 cm1 band, characteristic of CH3 bending modes, or

FIG. 6.2 Map of the birch bark harvesting areas in Romania: Aninei Mountains, Banat region (upper left) and Bucovina area (upper right). Bottom: typical birch bark appearance and exfoliated samples.

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FIG. 6.3 Typical data extracted from our FT-Raman spectral database of birch bark collections from the Apuseni Mountains, (single site) showing images of the outer raw bark (numbered from 1 to 9) and their characteristic FT-Raman spectra. Note the variability of the relative intensity of the bands in the 1601–1633 cm1 range. Excitation: 1064 nm, 350 mW. Vertical offset was applied for clarity. Adapted from S. Cinta-Pinzaru, C.A Dehelean, C. Soica, M. Culea, F. Borcan, Chem. Cent. J. 6 (2012) 67. PMCID: PMC3410787, Copyright ©2012 Cinta-Pinzaru et al.; license Chemistry Central Ltd. (CC BY 2.0) and from M. Steiner, S. Cıˆnta˘ Pıˆnzaru, Assessment of the betulin content in the Betula pendula Roth birch bark from Romanian flora, in: K. Nagy-Pora, V. Chis, N.S. Astilean, O. Cozar (Eds.), Book of Abstracts, 31st European Congress on Molecular Spectroscopy (EUCMOS), 2012, Napoca Star, Cluj-Napoca, Romania, 2012, p. 341.

the typical band at 1196 cm1 attributable to d(OH) and τ(CH2) bending modes (see Table 6.1). The high-wavenumber region of the Raman spectra from raw bark shows an envelope of multiple strong bands observed in the spectra of pure betulin and its derivatives (Table 6.1). FT-Raman spectra of raw birch bark harvested from various trees in the same geographical area displayed the same main bands, with notable differences in relative intensities as well as background intensity, the latter being associated with the presence of fluorescent compounds in the bark. Taking a closer look at the 1600–1650 cm1 spectral range, characteristic differences were observed for each bark sample. This variation appeared dependent on many factors such as the age of the tree, exposure to the sun, the soil, and other geoclimatic conditions (not given here). According to previous studies [46], and the vibrational assignment of betulin [47] and betulinic acid [48], the Raman band at 1601 cm1 is absent from the spectra of triterpenes of the lupane group. Therefore the band at 1601 cm1 could suggest other wood components, most probably associated to the presence of lignin. The relative

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FIG. 6.4 Typical FT-Raman spectra (800–3100 cm1 range) collected from raw birch bark samples (Betula pendula Roth) harvested from the spontaneous flora of three sites in the Bucovina area, Romania. The FT-Raman signal of solid, polycrystalline betulin (lup-20(29)-ene-3b,28diol,C30H50O2, Sigma Aldrich) is shown in the bottom part of the figure (black spectrum). Excitation: 1064 nm, 350 mW. Bark spectra collected from samples harvested from different locations encoded DN—Black Hill, DR—Runc Hill, and VD-Park—Vatra Dornei are shown in different colors, as indicated. The spectra are baseline corrected and vertically separated for better visualization.

intensity ratio R ¼ I1633/I1601 of the Raman bands at 1633 and 1601 cm1, the first being representative for triterpenes [16,46–48] and the second assigned to lignin, varied from 0.9 to 1.1 (
0.04) and indicated different amounts of triterpene content relative to the whole wood bark (Fig. 6.5). Raman spectroscopy data of raw birch bark revealed characteristic but slightly different features from one tree to another, with particular specificity in the relative intensity of the main bands in the 1600–1640 cm1 spectral range (Figs. 6.3 and 6.4), which was proposed as the Raman indicator of triterpene content [45]. The raw bark spectral data were further used as reference materials for natural extract yield evaluation. FT-Raman spectroscopy proved to be a practical technique for the prediction of the highest triterpene content in bark species from forestry areas, for the informed choice of harvesting times and places, and for the identification of individual genotypes directly in the field with the use of appropriate, market available, portable Raman instruments [45]. Moreover, no sample preparation was required and the analyzed bark could be further used for extraction protocols.

TABLE 6.1 Calculated Vibrational Wavenumbers at the B3LYP Level of Theory, Experimentally Observed Raman Bands of Raw Bark, Betulin, Betulinic Acid, Lupeol, and Ursolic Acid, Together With Vibrational Assignments Experimental Raman Data (cm21) Theoretical Data (cm21) [30]

Raw Birch Bark

Betulin

Betulinic Acid

Lupeol

Ursolic Acid

Vibrational Assignment

2970

2925 vvs

2927 vvs

2940 vvs

2936 vvs

2935, 2896 vvs

n(CH)

2906

2854 sh

2866 sh

2867 sh

2871 sh

2870 sh

n(CH2)

1716 m

n(C]O)

1658 s

n(C]C), d(CH2)

1710 w 1663

1633 s

1681, 1718 w 1642

1646 s

1643 s

1601 s

n(C]C) lignin

1480

1481 sh

1484 sh

1485 sh

1483 sh

1485 sh

d(CH2, CH3)

1462

1465 sh

1464 sh

1466 sh

1455, 1468 sh

1454 s

d(CH2, CH3)

1458

1440 s

1440 s

1442 s

1441 s

1432, 1442 sh

d(CH2, CH3)

1180

1195 m

1195 s

1195 s

1195 s

1201 w

d(OH), τ(CH2)

742

729 m

730 s

730 s

729 s

729 s

n(C–C) ring, r(CH3,CH2)

702

701 m

701 s

702 s

702 s

678 s

τ(CH2) terminal group + n(C–C)

d, bending; τ, twisting; m, medium; s, strong; n, stretching; vs, very strong; w, weak. Adapted from A. Falamas, S. Cinta Pinzaru, C.A. Dehelean, C.I. Peev, C. Soica, J. Raman Spectrosc. 42 (2011) 97–107; S. Cinta-Pinzaru, C.A. Dehelean, C. Soica, M. Culea, F. Borcan, Chem. Cent. J. 6 (2012) 67.

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Raman ratio R = I1633 /I1601

1.2

1.1

1.0

0.9

0.8 T T T T T T T T T T T T UT IN OU IN OU INT OU IN OU IN OU IN OU IN 1 O R 1 R 2 R 2 RK RK N 1 N 1 N 3 N 3 N 4 N 4 N 2 N 2 D D D PA PA D D D D D D DR D D

Place of harvesting

FIG. 6.5 Plot of the Raman relative intensity ratio of the triterpene and nontriterpene bands from spectra of raw birch bark samples (Betula pendula Roth) as a function of place of harvest. Mean, normalized, background subtracted spectra were employed. The place of harvesting (Bucovina area, northern Romania) is encoded according to the local names (DN—Black Hill, DR—Runc Hill, Park—Vatra Dornei; INT—inner side of the bark; OUT—outer side of the bark). Excitation: 1064 nm, 350 mW.

Currently it is generally accepted that the outer bark of the birch tree is rich in pentacyclic triterpene compounds such as betulin (B, lup-20(29)ene-3b,28-diol), lupeol (L, lup-20(29)-en-3-ol), and other minor components, such as betulinic acid (BA, 3b-hydroxy-20(19)-lupaen-28-oic acid), oleanolic acid, ursolic acid, and betulinic aldehyde [6]. According to data from recent literature, betulin content is dominant in the natural extract products from birch bark, while betulinic acid content is much lower [15,41,43,44,49]. Fig. 6.6 shows the FT-Raman spectra characteristic of standard triterpenes (betulin, betulinic acid, ursolic acid, and lupeol) compared with the spectrum from a raw bark specimen in the 1400–1800 cm1 spectral range (Fig. 6.6A) and the 2800–300 cm1 spectral range (Fig. 6.6B), respectively. The characteristic Raman signal of the reference compounds and their molecular structure is presented in Fig. 6.7. Complete vibrational characterization of the FT-Raman and FT-IR modes of betulin [47] and betulinic acid [48] were previously reported by our group. Vibrational data are of significant importance in identifying betulin and betulinic acid from other components found in bark extracts or their pharmaceutical formulations. To achieve a reliable assignment of Raman and IR bands observed experimentally, and for a detailed

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FIG. 6.6 FT-Raman spectra of the raw bark of the birch tree, betulin, betulinic acid, lupeol, and ursolic acid in the 1400–1800 cm1 (A) and 2800–3200 cm1 range (B), respectively.

understanding of the molecular geometry and structure, a comparison with theoretical spectra obtained using quantum chemical calculations, namely, density functional theory (DFT), was employed. The computed vibrational wavenumbers along with their IR intensities and Raman activities were used to identify the vibrational modes explicitly. A detailed vibrational assignment of the fundamental modes of betulin has been reported [47]. Based on this vibrational characterization, the 1642 cm1 band was assigned to a complex contribution of C]C stretching and CH2 bending vibrations in the terminal methyl group. The betulin-intense bands at 1484, 1464, and 1440 cm1 were assigned to CH2 and CH3 bending modes and the 1195 cm1 band was attributed to OH bending and CH2 twisting vibrations. Additionally, the intense band at 701 cm1 was assigned to twisting vibrations in the terminal group of triterpenoids (Table 6.1). Extensive studies reported on the investigated structural modification of betulin and betulinic acid and identified many derivatives with excellent biological activities, such as anti-HIV and antitumor [50,51]. The presence of hydroxyl or carboxyl groups at the C28 position of the lupane skeleton molecule is important in differentiating between betulin and other natural triterpenoids, like betulinic acid and lupeol. Recio et al. [52] showed that the skeleton structure had no critical influence on betulin’s antiinflammatory activity, but the presence of a hydroxyl group was of the highest importance when the compound was tested on edema, proving its remarkable bioactivity against this disease [52]. Mukherjee et al. revealed that C28 carboxylic acid in betulinic acid was essential for cytotoxicity [32]. Other studies have indicated that modifications made to betulin at the hydroxyl position produces agents that are effective against influenza-A and herpes simplex type-1 viruses, by evaluating the antiviral activity of in vitro assays [53]. Considering the

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FIG. 6.7 FT-Raman signal of the main pentacyclic triterpenes from birch bark (ursolic acid, lupeol, betulinic acid, and betulin, as indicated) showing the 0–1900 cm1 fingerprint range, along with their corresponding molecular structures. Excitation: 1064 nm, 350 mW.

biological importance of these chemical groups, our work paid special attention to assigning corresponding vibrational Raman bands [47]. The main triterpenes found in birch bark, i.e., betulin, betulinic acid, and lupeol, have a similar lupane skeleton structure, the difference being their characteristic functional groups: –COOH in betulinic acid; –CH2–OH in betulin; and –CH3 in lupeol. Ursolic acid presents a similar elemental composition to betulinic acid

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(C30H48O3), but lacks its pentacyclic ring, having five fused hexa-rings, as shown in Fig. 6.7. These differences are reflected in their characteristic Raman spectra (Figs. 6.6 and 6.7). For example, betulinic acid can be unambiguously discriminated from betulin or lupeol due to the presence of specific carboxyl bands at 1681 and 1718 cm1 (Figs. 6.6 and 6.7). Beside the strong Raman band at 1601 cm1, discussed in the previous paragraphs, the FT-Raman spectrum of the birch bark presents other particular bands in the 1710–1600 cm1 spectral range. A weak band observed at about 1710 cm1 in the bark signal could be tentatively assigned to the joint contribution of ursolic and betulinic acid to the overall spectral feature. Betulinic acid exhibits specific weak Raman bands (IR strong) at 1718 and 1681 cm1, whereas ursolic acid has a fingerprint band at 1718 cm1. These fingerprint bands, however, are less discernable individually in the spectrum of the bark due to a lower content of betulinic or ursolic acid. The C]C Raman mode of triterpenes is located at 1642 cm1 and at 1658 cm1 in the case of ursolic acid, whereas bark shows a strong, broad band centered at 1633 cm1, comprising a shoulder component at 1642 cm1. A possible explanation of the broad band at 1633 cm1 could be the presence of other aromatic compound(s), which are responsible for the “nontriterpene” band at 1601 cm1. The FT-Raman spectrum of the bark shows one intense band located at 1442 cm1, while the triterpenes present in this spectral region form a broad band with three peaks located at 1442, 1465, and 1481 cm1. Ursolic acid, however, shows a strong Raman fingerprint band at 1455 cm1, assigned to methyl deformation modes. The band observed at 1193 cm1 in the FT-Raman spectrum of the birch bark could be associated to the 1195 cm1 mode in the spectra characteristic to triterpenes, and is observed mostly in the spectra of lupeol and betulin. This vibrational characterization suggests that the main Raman spectral feature of the bark is dominated by the signature of betulin, highlighting its dominant content. IR spectroscopy has also been used for the identification of the main compounds found in birch bark, and allows their differentiation despite their high structural similarity. The IR band shape in the 1032–1006 cm1 spectral range of the bark (see Ref. [45]) confirms the triterpene contribution, since betulin was responsible for a band at 1006 cm1, whereas betulinic acid and lupeol exhibited a band at 1032 cm1. Moreover, betulinic acid exhibited a strong characteristic IR band at 1681 cm1 that allows fast differentiation among triterpene species [48]. When analyzing the content of each triterpene in the raw bark, however, the fingerprint band of betulinic acid was not distinctly observable in the FT-IR spectrum of the bark. The FT-IR results suggest that the contribution of betulinic acid is minor, being consistent with the findings from FT-Raman analysis and further validated by GC–MS [45]. The high wavenumber range of the IR spectra did not allow further differentiation of the bark components since the IR contribution of the bark presents an envelope of triterpene-specific spectral features in this range.

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BIRCH BARK EXTRACTION PRODUCTS: RAMAN SPECTROSCOPY ANALYSIS Natural extracts have received increased interest due to the fact that they can be employed therapeutically. The literature in this field presents various procedures for obtaining extraction products from the outer layers of birch bark, such as ultrasonic-assisted extraction [54], supercritical carbon dioxide extraction [55], or even green methods such as subcritical water extraction [56]. Studies have shown that the content of active components in birch bark differs between species. Diouf et al. [57] reported a 56% betulin content in the bark of yellow birch (Betula alleghaniensis Britton) from Quebec, Canada, while J€ager et al. [30] reported a 34% betulin content. A recent study showed the possibility of obtaining birch tree extract from outer bark comprising at least 70% betulin [58]. The green method for extracting betulinic acid reported by Liu et al. [56] found that under optimal conditions, the maximum content of extracted betulinic acid was 28.03 mg/10 g birch bark, higher than in previously reported methods (leaching extraction, reflux extraction, or ultrasonic extraction). Considering the high demand for pure triterpenes extracted from birch bark, Siman et al. developed a method for the purification of betulin based on the differential solubility of extract components in various solvents along with their crystallization or precipitation [59]. The purity of betulin using this method was 99%—at minimal cost. Our group’s contribution to this field was to identify the optimum solvent to use for a maximum extraction yield of betulin . We employed Raman spectroscopy to probe any spectral correlations with the content of the extraction products, assuming that the Raman ratio of the bands characteristic to the main compound relative to nontriterpene compounds depends on the extraction yield for a certain solvent. The obtained extracts were reported using various procedures [15,16,45]. The best results in terms of betulin quantity were obtained when we combined solvent extraction with heating procedures. This approach aimed to prepare a precipitate based on betulin’s sublimation property. A combination of procedures led to a content of over 90% betulin as a dry extract [60]. Thus unique improvements in the extraction procedures of betulin and betulinic acid from birch bark resulted in the production of a greater amount of active compound (over 90%) than previously reported. Soica et al. [15] employed several solvents for the extraction procedure: ethyl acetate, chloroform/dichlormethane/methanol (1:1:1), and dichloromethane. Further purification in n-hexane followed, depending on the employed solvent, and various amounts of betulin and betulinic acid were detected. The highest yield for betulinic acid was obtained with chloroform or dichloromethane, whereas betulin was best extracted with dichloromethane. The two-step extraction with a degreasing process did not change the betulinic acid quantity very much. However, in the case of betulin it may be more important as demonstrated by a reduced total extract volume. The ratio

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between the two main compounds, betulin and betulinic acid, in chloroform, dichloromethane, and methanol extracts, respectively, was found to be around 10:1, a value close to that found in the literature [61]. Zhao et al. simultaneously extracted betulin and betulinic acid from white birch bark using HPLC and different solvents, such as dichloromethane, ethyl acetate, acetone, chloroform, methanol, and ethanol. They found that the highest content of triterpene was obtained with 95% ethanol, and identified significant variations in the betulin and betulinic acid content in white birch bark growing in different locations in China [44]. When Soica et al. [15] used ethyl acetate, however, betulin was less extractable and a smaller ratio (7:1) was obtained. In another study by our group three different extraction methods were used alongside FT-vibrational spectroscopy and GC–MS data [45]. This study resulted in a ratio higher than 8:1. The highest triterpene content was obtained with methanol and longer extraction times, revealing betulin (59%) and lupeol (41%) as the two main components. A brief literature review on the various extraction methods of betulin using birch bark species as a raw material is given in Table 6.2. FT-vibrational spectroscopy was successfully applied for the identification of characteristic spectral features of skeletal triterpene modes in extracts. Kovac-Besovic et al. [66] identified betulin, betulinic acid, and lupeol in extracts from birch bark using IR spectroscopy based on their fingerprint IR absorption bands. In a preliminary study, our group combined for the first time FT-IR and FT-Raman techniques for the assessment of dry extracts of birch tree leaves, buds, and bark in order to monitor the presence of pentacyclic triterpenes [16]. Using FT-IR spectroscopy, the presence of acid species could be easily monitored in extract samples, based on the vibrational mode of the carboxylic group at 1684 cm1, which is absent in the IR spectrum of betulin. IR bands characteristic to betulin were dominant, suggesting its higher content, in agreement with the findings of conventional chromatography techniques. Two examples of extract evaluation using Raman spectroscopy are described below. 1. FT-Raman spectra collected from vegetal extracts obtained following similar protocols but using different solvents are presented in Fig. 6.8. They were compared with a reference spectrum of betulinic acid and betulin. The natural extracts showed similar vibrational behavior and the small differences observed from one spectrum to another can be attributed to the presence of molecular species other than betulin and betulinic acid, used here as references. All extraction products show high-intensity Raman signals with well-resolved features. The main bands reproduced in all the spectra are 1640 cm1, 1605 cm1, the group of bands in the 1400–1500 cm1 spectral region, and the 1195, 1018, 942, 913, 732, and 700 cm1 bands. Similarly, with the case of raw bark spectra, discussed in earlier sections of this chapter, the band at 1605 cm1 is assigned to other components than

TABLE 6.2 Review of Birch Bark Extracts Reported Using Various Birch Species, Extraction Methods and Solvents, and Their Pharmacological Effects Betulin Content in Birch Bark Extract

Vegetal Source

Method

Pharmacological Effects

References

90% Betulin

White birch Betula pendula Roth, Aninei Mountains, Romania

Soxhlet extraction with organic solvents (ethanol and propanol)

Antiproliferative effect on A431 (skin epidermoid carcinoma), A2780 (ovarian carcinoma), HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma); antiinflammatory effect on TPAinduced mouse model of ear inflammation

Dehelean et al. [60]

4.8 mg/g Dry extract—wood; 10.2 mg/g dry extract; 9.9 mg/g dry extract—foliage

Yellow birch wood, bark, and foliage Betula alleghaniensis Britton, Quebec, Canada

Maceration in 95% ethanol

– The foliage extract had the most potent radical scavenging capacity while wood and twig extracts exerted the highest inhibitory effects on the production of NO in LPS/INF-stimulated RAW 264.7 macrophages

Diouf et al. [57]

Ultrasonic-assisted extraction (UAE) in 95% ethanol

Extracts obtained by UAE present lower cytotoxic activity on RAW cells

5.6 mg/g Dry extract—wood; 9.2 mg/g dry extract; 4.2 mg/g dry extract— foliage

Continued

TABLE 6.2 Review of Birch Bark Extracts Reported Using Various Birch Species, Extraction Methods and Solvents, and Their Pharmacological Effects—Cont’d Betulin Content in Birch Bark Extract

Vegetal Source

Method

Pharmacological Effects

References

Approx. 80 mg/100 g

Betula alba cortex, Germany

Accelerated solvent extraction with n-hexane

– The extract is safe to be administered for in vivo administration animals and could be further used for pharmaceutical and pharmacological research

J€ager et al. [30]

57% Betulin

Betula pendula Roth, Aninei Mountains, Romania

Soxhlet extraction with chloroform/dichloromethane/ methanol (1:1:1) solvent mixture

Cytotoxic effects against A431 (skin epidermoid carcinoma), HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) comparable with betulin and betulinic acid

Soica et al. [15]

58.8% Betulin

Betula pendula Roth, Aninei Mountains, Romania

Soxhlet extraction with methanol

Not considered for studies of pharmaceutical effects

Cinta-Pinzaru et al. [45]

Cytotoxicity toward human gastric carcinoma (EPG85–257) and human pancreatic carcinoma (EPP85–181) drug-sensitive and drug-resistant cell lines

Drag et al. [62]

22.8% Betulin

Soxhlet extraction with chloroform/dichloromethane/ methanol (1:1:1) solvent mixture

23.7% Betulin

Ultrasonication bath followed by Soxhlet extraction with methanol

91% Betulin

Betula pendula Roth, Wroclaw (Lower Silesia), Poland

Rotary evaporator in ethanol (100%)

75.4% Betulin

Betula pendula Roth, Betula pubescens Ehrh., Estonian veneer industry

Continuous extraction procedure with n-heptane including clarification crystallization

Low toxic behavior in RC-37 cells—African green monkey kidney cells; antiherpetic activity in vitro; highly active against acyclovir-resistant herpes virus strains

Heidary Navid et al, [63]

75% Betulin

Birch bark extract as hard gelatin capsules containing 20 mg of dry alcohol extract from birch bark per capsule, SNS—Pharma, St. Petersburg, Russia

—

Beneficial effects of a 12-week treatment, for patients with chronic hepatitis C infection, with birch bark extract included reduced fatigue, reduced abdominal discomfort, reduced depression, reduced dyspepsia

Shikov et al. [64]

Not specified

Betula utilis, Himalayan silver birch, Betulaceae, Govind Wildlife Sanctuary, Uttarakhand, India

Dried bark was milled to powder and extracted with methanol, evaporated, and dried. The methanol extract was suspended in water and extracted successively with n-hexane, chloroform, ethyl acetate, and n-butanol

All extracts were tested for in vitro cytotoxic activity against nine different human cancer cell lines (A172—glioblastoma, MCF-7—breast adenocarcinoma, DLD1—colorectal adenocarcinoma, PLC/PRF/5—liver hepatoma, A549—lung carcinoma, SK-OV-3—ovarian carcinoma, BxPC-3—pancreatic adenocarcinoma, DU145—prostate carcinoma, and Caki-1—renal carcinoma). The ethyl acetate extract proved to be the most active compared with other extracts

Mishra et al. [65]

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FIG. 6.8 FT-Raman spectra of the betulin-based extracts in various solvents (e2–e6) compared with the spectra of betulin and betulinic acid; e2—ethyl acetate extract, e3—chloroform extract, e4—dichloromethane extract, e5—methanol extract, and e6—dichloromethane extract.

triterpenes (most probably lignin) which are present regardless of the extraction protocol. In the high-wavenumber region, the extracts exhibit a very strong band with four peaks located at 2972, 2930, 2913, and 2869 cm1. The Raman bands observed in the spectra of the extraction products follow the envelope of reference samples with maximum at 2930 cm1 centered on the Raman band of betulin species. However, due to the large overlap, this spectral range does not allow differentiation of the triterpene species. Betulinic acid was less observable in the spectra of the extracts due to the weak Raman intensity characteristic of the –COOH group at 1681 and 1718 cm1, and the betulin spectral Raman signature was dominant in the Raman spectra of the extracts, confirming the presence of betulin as the dominant species in all the extraction products. The calculated Raman ratio between the triterpene and nontriterpene contributions to the overall spectral feature of the two key bands at 1640 and 1605 cm1 resulted in a rapid hierarchy of extract quality: (e6) showed a factor of 1.22, (e5) 0.88, (e4) 0.94; (e3) 0.99, and (e2) 0.98, respectively. Thus the (e6) extract in dichloromethane resulted in the best extraction efficiency, further confirmed by conventional methods [11,29]. To conclude, Raman spectroscopy can provide quantitative information regarding the triterpene content in the extraction products by analyzing the relative

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intensity ratios between the 1640 cm1 band, assigned to the C]C mode of triterpenes, and the 1600–1606 cm1 band, assigned to nontriterpene components—also evidenced in the Raman spectra of the bark. 2. Another method for obtaining natural extracts and for their characterization using vibrational spectroscopy involved six natural extract samples (denoted 1–4, 1 pp, and 3 pp) obtained from various amounts of dry birch bark which underwent 6 h of extraction either in 400 mL of 2-propanol (samples 1 pp and 3 pp) or 400 mL of ethanol (96%) (samples 1–4) [60]. Thus the dried weight of the starting material was 9.96 mg (1), 8.08 mg (2), 10.31 mg (3), 8.12 mg (4), 10.47 mg (1 pp), and 12.05 mg (3 pp), respectively. The final appearance of the extract was a cream-colored solution with white crystals for precipitate samples. The FT-Raman spectra of these extract samples are presented in Fig. 6.9. The highest quality spectra were seen for samples encoded 1 pp and 3 pp. These two samples also showed the highest purity, which correlated with the lowest intensity 1606 cm1 band seen in all the spectra. On the other hand, extract sample 3 presented the worst signal and thus the lowest triterpene level. The relative intensity ratio R ¼ I1642/I1606 was calculated for all the spectra in Fig. 6.9 and the data compared with results obtained from HPLC measurements thereby indicating the betulin content in each extraction product (Fig. 6.10). The comparative plot from Fig. 6.10 indicates a good correlation for the five extracts (out of six) and their classical content determination is described in Ref. [60]. The Raman and HPLC data follow the same trend with a discrepancy for only one of the samples. This could be related to the Raman background associated with fluorescence impurities, which is usually observed in spectra collected from vegetal tissue. Equally, it could be related to the errors associated with the multiple steps involved with liquid chromatography determination. In any case, the results indicate the capability of Raman spectroscopy at identifying the main active compounds in the obtained natural extracts as well as its potential to become a valuable analytical tool for natural product research. The high volumes of betulin obtained using this extraction protocol makes the procedure attractive in terms of large-scale extraction using underestimated and underexploited natural birch bark as a resource. These results indicate that Raman spectroscopy could be successfully employed for use as the standardization procedure of birch tree extracts, considering its high selectivity, lack of any requirements for sample preparation, and its nondestructive character. Bearing in mind the possible antitumor activities of these compounds, natural extracts show great promise in terms of obtaining pharmaceutical derivatives from birch bark because the procedure is relatively simple and relatively cheap.

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FIG. 6.9 FT-Raman spectra of six extracts compared with the FT-Raman spectrum of betulin (the code samples (1–4), 1 pp, and 3 pp are shown on each spectrum). A high-quality extract (high betulin content) is associated with the low intensity of the band in the 1600–1606 cm1 range.

FIG. 6.10 Comparative plot to validate the Raman data (relative intensity ratio R ¼ I1642/I1606) for extracts described in Fig. 6.9 by comparison with their HPLC data (betulin content given in milligrams).

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PHARMACEUTICAL FORMULATION OF BETULIN AND BIRCH BARK EXTRACTS Ongoing research is focused on the effectiveness of pentacyclic triterpenes in skin pathologies such as melanoma and skin cancer, with their activity being under exhaustive biomedical and pharmaceutical consideration. Therefore special attention is currently being paid to the possibility of creating pharmaceutical formulations based on betulin and its metabolic successor betulinic acid for testing on skin malignancies and other diseases. The process of drug discovery from plants represents a complex chain of events involving several steps, such as plant collection, preparation of plant-based extracts, the bioassay of extracts, and the isolation and elucidation of the active components of a particular structure. Subsequent steps include production of formulations, toxicity studies, delivery mechanisms, and activity studies (Fig. 6.11). The major problem encountered when trying to formulate pharmaceutical compounds from these triterpenes is their low hydrosolubility. In this manner, several possible solutions have been proposed and investigated, such as inclusions based on pentacyclic triterpene–cyclodextrin guest–host-type complexes, triterpene-based nanoemulsions, ointments, structural modifications [21], and liposome-based or glycoside-based formulations [67]. Various structural modifications of triterpenes have been investigated with the aim of estimating their inhibitory activity on cancer cell growth [50].

FIG. 6.11 Graphical sketch showing the steps conducted in our research to obtain betulin-based products—from main ingredient identification to extraction, analysis, and various formulations. Cyclodextrin excapsulation image component is adapted from A. Falamas, S. Cinta Pinzaru, V. Chis, C. Dehelean, J. Mol. Str. 993 (2011) 297–301 and nanoemulsion image from S. Cıˆnta˘ Pıˆnzaru, C.A. Dehelean, A. Fa˘la˘mas, M. Steiner, S. Ganta, M. Amiji, V. Chis, in: Beat Loffler, Patrick Hunzicker (Eds.), Proceedings of the CLINAM-8/2015—The European Summit for Clinical Nanomedicine and Targeted Medicine, European Foundation for Clinical Nanomedicine. Basel, Switzerland, 2015, p. 119.

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Mukherjee et al. [32] observed that structural modifications at the C2 position in betulinic acid enhanced cytotoxicity. Additionally, they observed that the three rings forming the skeleton (the A, B, and C rings) played an important role in anticancer activity, suggesting that these modifications could pave the way for the design of new anticancer drugs [32]. Alakurtti et al. [4] presented an excellent review of structure–activity relationship studies and the pharmacological properties of betulin derivatives, such as their antitumor, anti-HIV, and antiinflammatory properties [4]. Betulin derivatives act by triggering cell death pathways, such as apoptosis or programmed cancer cell death, without harming normal cells, making their use preferable to chemotherapeutic agents [68]. Ionic derivatives exhibited higher water solubility thus showing much higher inhibitory effects against different cancer cell lines [68]. Other methods for improving the solubility of triterpenes and their bioavailability consist of incorporating an active agent in liposomes. Liposomes are a promising alternative to conventional drug delivery systems as they can encapsulate both hydrophilic and hydrophobic materials and exhibit good biocompatibility, biodegradability, low toxicity, and controlled drug release [69,70]. Betulinic acid, encapsulated in a lipid bilayer of liposomes, covered by a polyethylene glycol layer would result in a 142-nm structure which could easily accumulate in tumor tissues [71]. This new formulation displays an improved tumor inhibitory effect compared with that of free betulinic acid in both in vitro and in vivo experiments. Betulinic acid incorporated in liposomes and intravenously delivered to nude mice with human colon and lung cancer tumors reduced tumor growth by more than 50%, leading to an enhanced survival rate in the mice [72]. Cyclodextrins (CDs) are torus-like molecules formed of glucopyranose units. In aqueous solutions, CD cavity is occupied by high-enthalpy water molecules, which can be easily substituted with drug molecules. Following molecular encapsulation, the drug dissolves more rapidly, has a better solubility limit, and results in more complete absorption. The release of drug molecules from an inclusion complex is assisted by their replacement with appropriate molecules, such as lipids. Alternatively, the drug may be transferred to a system for which it shows higher affinity [73,74]. Our research group has investigated the possibility of betulin encapsulation into CDs using two preparation methods based on physical mixing and kneading of 1:1 and 1:2 M ratios of betulin: hydroxy-propyl-gamma-cyclodextrin (HPGCD) [27]. The guest–host-type compounds, as well as the two separate molecules, were analyzed by quantum chemical calculations and FT-Raman spectroscopy. A detailed vibrational analysis could help us understand the mode of interaction between betulin and cyclodextrin molecules when formulating pharmaceutical compounds. The FT-Raman spectra of the inclusion complexes exhibit the main characteristic features of HPGCD and to some extent additional contributions from the guest species (Fig. 6.12). The Raman spectrum of the 1:1 M inclusion complex resembles well the sum of the individual

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FIG. 6.12 SEM pictures of betulin (A), HPGCD (B), and their inclusion complex (C). FT-Raman spectra of betulin (a), HPGCD (b), 1:1 and 1:2 M inclusion complexes (c and d, respectively). Spectra adapted from A. Falamas, S. Cinta Pinzaru, C.A. Dehelean, C.I. Peev, C. Soica, J. Raman Spectrosc. 42 (2011) 97–107, Copyright (2011), with permission from John Wiley & Sons, Ltd.

spectra of the guest and host molecules, while the Raman spectrum of the 1:2 M inclusion complex shows a profile that is more similar to the spectrum characteristics of HPGCD. The vibrational analysis presented in this study suggests an interaction between betulin and cyclodextrin. A broadening, a decrease in intensity, and shifts in wavenumbers of betulin characteristic vibrational modes, clearly visible in the FT-Raman spectra of the inclusion complexes, confirms the formation of inclusion complexes. The vibrational analysis suggests an interaction between betulin and cyclodextrin through betulin’s alkene moiety group, leaving the cyclodextrin cavity of the CH2OH group outside the betulin molecule, allowing it to remain available for further interactions. Moreover, in the case of the 1:2 M inclusion complex, the vibrational modes assigned to the alkene moiety group of betulin decrease in intensity, suggesting better encapsulation of the betulin molecule, possibly due to

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better shielding of betulin by the two HPGCD molecules. Thus the Raman signal for the inclusion complex of the 1:2 ratio better resembles the cyclodextrin signal. Additionally, the betulin band at 1642 cm1 has a weaker Raman intensity compared with the 1:1 complex [27]. The formation of the inclusion complex was sustained by analyzing scanning electron microscopy (SEM) images of betulin, HPGCD, and their 1:1 M inclusion complex. The shape and surface morphology are depicted in Fig. 6.13. Betulin has a specific shape comprising of needle-like crystals of different sizes with smooth surfaces. The cyclodextrin is composed of spherical, smooth, broken particles. The SEM image of the complex shows irregular prismatic crystals of different shapes and sizes. The complex presents important morphology changes from its initial substances, a characteristic which strongly suggests complexation [75]. The inclusion of betulinic acid in CDs also revealed increased water solubility. SEM images of the physical mixture of BA and GCDG (not shown here) showed both crystalline and amorphous particles adhering to mutual surfaces exhibiting small particles with a clear tendency to aggregate, a specific behavior of amorphous particles comprised of a single component—suggesting real complexing took place [76]. The inclusion complexes were applied in vitro and in vivo on melanoma tumor models in mice and led to a visible reduction in tumor dimensions [76]. Despite the advantages offered by cyclodextrin inclusion complexes, it is generally believed that CDs find it difficult to penetrate intact skin, a situation which might hamper triterpene active drug delivery for skin injury treatments. For this reason, a second method for improving solubility was tested. Nanoemulsions (NE) are widely known as efficient drug carriers [77]. They are dispersions of oil and water stabilized by an interfacial film of droplets with sizes of less than 500 nm. Nanoemulsions are composed of biocompatible phospholipids as emulsifiers and they can incorporate significant amounts of drugs in the high-volume fractions of their oil cores. In terms of the release rates of active ingredients in pharmaceutical compounds, a key parameter is the size of the particles—the smaller the particles, the higher the release rate. Some of the advantages offered by nanoemulsions are: (1) they have no flocculation or sedimentation; (2) they can be formulated as foams, liquids, or sprays; (3) they are nontoxic; and (4) they are nonirritant. Thus they are considered suitable for human and veterinary use. Additionally, they are easy to scale up, they are cost effective, and relatively stable when compared to many other nanosized drug delivery systems like liposomes. Nanoemulsions have various applications in cosmetics, antimicrobial applications, or cancer therapies [78], all based on the fact that they offer transdermal delivery, targeted drug delivery, and improved delivery of poorly soluble drugs [79]. A betulin nanoemulsion was formulated and applied to mice models, with induced melanoma, to test antitumor activity [80,81]. The histopathological evaluations of skin confirmed differences between the betulin nanoemulsion treated and untreated mice groups, indicating that the betulin treatment offered a potent

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FIG. 6.13 Transmission electron microscopy images of a betulin-containing nanoemulsion (A) and a blank one (B). The diameter is indicated on each nanodroplet. Scale bar equals 100 nm. (C) FT-Raman spectra of betulin (a), botulin-based nanoemulsion (b), and blank nanoemulsion (c). Panel B: Image adapted from S. Cıˆnta˘ Pıˆnzaru, C.A. Dehelean, A. Fa˘la˘mas, M. Steiner, S. Ganta, M. Amiji, V. Chis, in: Beat Loffler, Patrick Hunzicker (Eds.), Proceedings of the CLINAM-8/2015—The European Summit for Clinical Nanomedicine and Targeted Medicine, European Foundation for Clinical Nanomedicine. Basel, Switzerland, 2015, 119. Panel (C): Adapted from A. Falamas, C.A. Dehelean, S. Cinta Pinzaru, Vib. Spectrom. 95 (2018) 44–50, Copyright (2018), with permission from Elsevier.

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reduction in skin tumor damage and inflammatory processes. Betulin’s solubility and bioavailability issues could be solved by incorporating it in a nanoemulsion formulation, which would enhance efficacy and lower toxicity. In this way, a formulation offering potent antiinflammatory and anticarcinogenic activity, alongside low skin toxicity, could be developed. Moreover, it could influence the penetration of carcinogens and reduce damage to the main organs like the liver [80]. The betulin nanoemulsions formulated by our group were further used in mice skin treatments with the effects investigated using micro-Raman spectroscopy [81]. Transmission electron microscopy (TEM) images show that a nanoemulsion is composed of spherical droplets with uniform distribution (Fig. 6.13 A and B). The average diameter of a nanoemulsion droplet is 200 nm, with zeta potential values of –23 mV for betulin nanoemulsions [80] and –39 mV for betulinic acid nanoemulsions [26]. The drop in charge in the case of the betulin nanoemulsion may be due to the partitioning of some betulin to the interface or into aqueous phase. Finally, measurements indicated that the encapsulation efficiency of both triterpenes was 90% [26,80]. For a complete characterization of blank and betulin-containing nanoemulsions, FT-Raman spectroscopy was employed. Fig. 6.13C presents the FT-Raman spectra of betulin powder (a), comparison with those of a betulin nanoemulsion formulation (b), and a blank emulsion (c). The FT-Raman signal of both nanoemulsions is dominated by the characteristic Raman bands of flaxseed oil (not shown here). The most intense peaks are assigned to ester C]O stretching mode at 1745 cm1, cis C]C stretching mode at 1656 cm1, CH2 scissoring mode at 1438 cm1, in-plane methylene twisting at 1297 cm1, and symmetric rocking in cis double bonds (]C–H) at 1265 cm1. The symmetric and asymmetric C–H stretching vibrations of methyl and methylene groups in the 2847–3007 cm1 range dominate the high-wavenumber region. The high degree of overlap between nanoemulsion Raman bands with those characteristic to betulin hamper its monitoring within a nanoemulsion formulation when using conventional Raman spectroscopy. Keeping in mind the encapsulation efficiency result of betulin in oil droplets, as well as the unfortunate overlapping of betulin fingerprint bands with those of flaxseed oil, we concluded that FT-Raman spectroscopy alone is less efficient at monitoring the active specimens in a nanoemulsion when betulin is of interest [81]. However, subtle differences could be used by employing chemometrics when discrimination is the research target. However, a vibrational characterization was possible and the FT-Raman technique evidenced the oil phase of the emulsion. The production rate of newly approved medicines has seen a serious decrease in the last number of years [82]. Potential for innovation relies heavily on research and development. Due to an increased incidence of cancer, the need for analytical methods to determine the effects of anticancer drugs has heightened. Natural compounds hold great promise for new and improved drugs, however, there are obstacles to overcome. The identification

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of natural compounds which can be used effectively against human diseases, toxicity, drug interactions with intracellular biomolecules, as well as the unpredictable pharmacokinetic properties are all important steps which need consideration prior to formulating a new drug.

EVALUATION OF BETULIN’S THERAPEUTIC EFFECT ON MICE SKIN BY MEANS OF RAMAN SPECTROSCOPY Betulin and its derivatives may exhibit different molecular effects which are structure-related. Molecular modifications at various positions (hydroxyl or alkene moiety groups) may increase or decrease solubility, binding ability, and medicinal properties. Therefore this represents the ideal starting point for semisynthetic derivatives with important biological activities, especially linked to cancer and HIV topics. Vibrational spectroscopy techniques have gained a lot of interest from the pharmaceutical and biomedical fields due to their recent advances. Raman spectroscopy provides nondestructive, rapid, biomolecular-relevant information, offering high chemical specificity due to the vibrational bands found in Raman spectra. The application of Raman spectroscopy in the field of pharmaceuticals has shown immense potential, having been successfully applied to the identification and quantification of active drugs and excipients in pharmaceutical formulations [83,84]. Moreover, cell-based biosensors, used for monitoring the interaction of cells with drugs, have been developed using micro-Raman spectroscopy [37]. In recent years, Raman spectroscopy has gained a great deal of interest in disease diagnosis, particularly cancer, because of its ability to provide data-rich, specific information about tissues [85]. Cancer in its earlier stages presents cells with an increased nucleus-to-cytoplasm ratio, deficiencies in their DNA structure, higher metabolic activity, and changes in lipid and protein levels, all of which differ from their healthy analogous. As recently reviewed by Kong et al. [40], Raman techniques have been successfully applied in biological screening for diagnostic purposes, compatible with clinical environments. Raman studies of both hard (bones and teeth) and soft tissues (epithelial, muscular, and nervous) from various organs, such as the lungs, breasts, brain, liver, or skin have been reported. Micro-Raman spectroscopy is suitable for cancer diagnosis because of its sensitivity in detecting these subtle biochemical changes. The molecular changes which occur in skin cancers [86] such as squamous cell carcinoma, malignant melanoma, and basal cell carcinoma have been evaluated using vibrational spectroscopy [87,88]. Accurate monitoring of clinical treatment is essential for quality of life, performance index, and the survival of patients. The clinical field is in great need of optical imaging techniques that can be used noninvasively and in a rapid manner. Thus it is crucial to develop sensitive techniques that can diagnose diseases and directly monitor treatment-induced modifications. The great appeal of

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micro-Raman spectroscopy, which has recently been applied in the biomedical field, lies especially in its potential for in vivo applications. Taking into account the promising biological activities of the main triterpenes extracted from birch bark, our accomplishment in applying Raman spectroscopy to investigating betulin-based compounds for various skin pathologies induced in mice specimens, and their therapeutic effects, are briefly discussed here. These experiments were part of a large multidisciplinary approach where reproducible melanoma was obtained in mice by UV exposure, through chemical promoters (TPA-DMBA), or through B16 melanoma cell injection. Extraction products as well as triterpene-based pharmaceutical formulations, having potential efficacy in treating skin cancers, were prepared and tested [89–93]. Skin and tumor samples were histologically analyzed and specific skin parameters, including transepidermal water loss (TEWL), melanin, and erythema, were measured by noninvasive methods. Our results indicated that triterpenetreated groups presented a smaller number of skin lesions and tumors compared with the control group. Moreover, the development and occurrence of skin tumors were delayed by treatment with betulin or betulinic acid. An improvement in skin parameters was also observed in the treated groups. All the observations lead to the conclusion that pentacyclic triterpenes play a protective role in terms of the skin and are active compounds against skin damage. The histopathological evaluations were correlated with in vivo and ex vivo confocal micro-Raman, as well as surface enhanced Raman scattering (SERS) investigations which were applied to study the molecular changes along malignancy and treatment of animal skin [93]. The Raman and SERS signals from tissues showed very good reproducibility and revealed clear differences associated with pharmaceutical treatment efficiency [91,92]. One experiment focused on the in vivo assessment of four animal models with different skin pathologies, investigated on the same day as pathology evolution [81]. The aim of the study was to use micro-Raman and SERS spectroscopy for the differentiation of skin pathologies and to monitor the effect of a betulin nanoemulsion topically applied treatment. In this manner, mice specimens were split into four groups as follows: the first group was treated with chemical carcinogens 7, 12 dimethylbenzanthracene (DMBA) as a cancer initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as cancer promoter; the second group was treated with the natural compound betulin, formulated into a nanoemulsion after cancer development; and the third and fourth groups were controls—one healthy and one treated only with the solvent used for dissolving the applied chemicals (Fig. 6.14). The appearance of skin cancer in the carcinogen-treated mouse model was confirmed by histopathological investigations (Fig. 6.14B), indicating that the time of carcinogen exposure was enough for the apparition of superficial pathology. A squamous carcinoma was correlated from the histopathological point of view with the damaged keratinocytes [94]. The H&E staining evaluations indicated tumoral areas (highlighted by the circle in Fig. 6.14B) had

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FIG. 6.14 (A) Macroscopic and (B) histological evaluation (H&E staining, original magnification 40) of mice treated with carcinogens (DMBA/TPA), mice treated with a betulin nanoemulsion, and mice in control groups. The circle in the H&E stain (row B) under the carcinogens application group indicates tumor development; presence of tumor proliferation and small cells disposed immediately subepithelial (Carcinogens application group); skin with hyperkeratosis and hyperplasia but absence of tumor cells or inflammation (Betulin nanoemulsion group); and skin with a normal histological aspect (Control group). (C) In vivo micro-Raman spectra characteristic of each group, collected from the surface skin of mice under anesthesia. Adapted from A. Falamas, C.A. Dehelean, S. Cinta Pinzaru, Vib. Spectrom. 95 (2018) 44–50, Copyright (2018) with permission from Elsevier.

developed in the group which experienced carcinogen applications. The betulin nanoemulsion applied on the skin had adequate rheological properties and a composition that determined a good delivery of betulin. Visual observations of the skin demonstrated an intervention by the active ingredient in skin recovery, and H&E staining indicated significantly reduced tumor growth when a betulin nanoemulsion was administered (Fig. 6.14B, the betulin nanoemulsion group). Betulin added in specific topical formulations was very active in terms of actinic keratosis [95]. The findings are well correlated with other investigations indicating betulin as an effective compound against lesions and severe skin damage [80]. It should be considered not only as a precursor to obtaining an active antitumor compound but a potent antitumor agent in its own right. These observations strongly suggest its prophylactic

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(application before carcinogen) properties, but also its ability as a curative agent (no sign of important damage). The H&E evaluations indicated that tumor development was only evident in the nontreated group. Our findings using micro-Raman spectroscopy exhibited direct evidence of molecular changes associated with nanoemulsion effectiveness (Figs. 6.14 and 6.15). The position of the major Raman bands characteristic to the control group were similar to the ones reported in the literature for healthy skin [96,97]. We identified specific biomolecular spectral markers sensitive to pathology evolution such as the amide III bands of collagen in the 1240–1300 cm1 spectral range and the 1300–1340 cm1 nucleic acids region. Other major Raman bands, such as the amino acid band, specific to collagen at 853 cm1, the 1448 cm1 vibrational band of lipids and proteins, the amide I band of proteins at 1655 cm1, and the CH2 symmetric and asymmetric stretching vibration bands in the 2850–2940 cm1 spectral area, were shifted to lower wavenumbers and showed a decrease in relative intensity in betulin nanoemulsion– treated skin and cancerous ones when compared to the control skin spectra. Plotted values of the calculated areas of bands sensitive to molecular change clearly showed differentiation in skin pathology, as highlighted in the insert to Fig. 6.15. The shift toward lower wavenumbers observed in cancerous skin, especially for the amide I band, indicated pathologically modified skin. The intensity decrease of the amide III bands in the 1240–1300 cm1 spectral range, together with the intensity increase of nucleic acid bands in the 1300–1350 cm1 spectral range, were also reported by other research groups [97], and could be considered as spectral biomarkers for cancerous skin (Fig. 6.15). The decreased intensity of collagen in the amide III and amide I spectral regions indicate a major loss of protein or changes in secondary protein structure. The same results were obtained in human skin subjected to strain [98]. On the other hand, the intensity gain of the nucleic acid bands in cancerous spectra could be related to an increase in nucleic acids in malignant tissues due to an elevated quantity of mitoses and increased duplication of genetic material responsible for the proliferation of malignant cells. The spectral changes indicated by Raman spectroscopy, and correlated with histopathology investigations, indicate the capability of the Raman technique for direct, in vivo differentiation of spectral changes related to various skin pathologies. Moreover, the betulin-based treatment efficiency reinforced the need for further studies in various formulations and delivery channels, suggesting great promise in terms of skin cancer. The betulintreated skin could be differentiated from other skin pathologies and the observed molecular changes supported the effectiveness of the betulin nanoemulsion formulation. Due to the small Raman cross-section and the strong autofluorescent background of biological media when excited with visible lasers, Raman spectroscopy has some limitations in trace bioanalysis. As a consequence, in the past

FIG. 6.15 (A) Averaged (from 30 spectra acquired from each skin pathology), in vivo Raman spectra collected from mouse skin treated with carcinogens (cancerous), skin treated with a betulin nanoemulsion (betulin treatment), and skin from control groups. Excitation: 785 nm. (B) The calculated band areas of the Raman biomarkers (1240–1300, 1300–1350, 1449, and 1656 cm1) characteristic to each skin pathology: control—black square; betulin treated—red circle; and cancerous—blue triangle.

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decades there has been a renewed interest in techniques to enhance the Raman scattering signal. One of the most popular is surface enhanced Raman spectroscopy (SERS), which provides huge amplification of the Raman signal of molecules found in the vicinity of nanostructured noble metal surfaces. Therefore the development of biocompatible nanosensors for the investigation of local chemical information on native biological molecules or nanovehicles that can target specific diseases and biocomponents in living systems is currently a major research focus [99–104]. Currently, there are two main SERS configurations used in biosensing: an intrinsic and an extrinsic route. The first one applies the analyte on the nanosurface and measures the Raman spectrum of the target biomolecule directly, while the latter one applies the specimen on nanoparticles using a Raman reporter whose signal is used for detection. Moreover, a complex structure can be formed and the sensor can be decorated with capture molecules such as antibodies, peptides, or small molecules. These nanosensors can be used for in vivo tumor targeting and cancer cell imaging [102,103]. The implementation of nanosensors in early disease detection, and subsequent treatment monitoring, may result in more successful patient outcomes and reduced treatment side effects. Our group expanded this concept for the first time to tissues, employing Ag colloidal nanoparticles incubated into different types of tissue samples [104], and demonstrated the possibility of recording high-quality, molecular specific SERS spectra. This trend has been since continued by other research groups using SERS for the study of biological tissues or for the differentiation of normal and cancerous biological samples [105–107]. We probed the SERS characterization of skin samples and its differentiation between normal and cancerous tissue and attempted to elucidate the SERS mechanism in biological tissue [81,92]. The SERS signal differed substantially from the well-known Raman signal of skin tissues (Fig. 6.16) and allowed differentiation of skin pathologies [81]. We were able to obtain a high-quality SERS signal which allowed us to evidence and differentiate the molecular components from tissues and draw conclusions about malignancy at a molecular level. The spectral signatures characteristic to healthy skin and melanoma-induced skin in mouse models, and the pathology evolution when a betulin nanoemulsion formulation was topically applied to the cancerous skin of mice, were investigated [81]. SERS markers associated to each pathology were identified and the signal was distinguished from the classical Raman signal of skin based on several biomarkers, such as the disappearance of the amide I band of proteins, the amplification of the 1574 cm1 band assigned to nucleic acid bases, and the appearance of the highly amplified band at 230 cm1 characteristic of metal– biomolecule complexes (Fig. 6.16) [81]. We also investigated the efficiency of Raman enhancement using Ag and Au colloidal nanoparticles labeled with cresyl violet Raman reporter molecules in cancer tissues [93]. Unlabeled Ag and Au nanoparticles were inoculated into ex vivo skin tissues via injection or by immersing the samples in

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FIG. 6.16 Raman spectrum (A) collected from a point at the surface of the skin and SERS spectra (B, C) collected from the Ag NPs injected skin layers. Inset shows a typical in vivo Raman spectrum acquired from mouse skin treated with betulin nanoemulsion before AgNPs injection. Y-axis magnification has been applied for clarity. Excitation 785 nm, 50 objective, 5 s integration time. Reproduced from A. Falamas, C.A. Dehelean, S. Cinta Pinzaru, Vib. Spectrom. 95 (2018) 44–50, Copyright 2018, with permission from Elsevier.

colloidal solutions. The results presented an enhanced Raman signal of tissue components in the vicinity of the nanostructures that had been taken up. Different signals were obtained for healthy and diseased samples. Furthermore, amino-functionalized nanoparticles were employed to record SERS signals from tissues [108]. Cresyl violet chemisorbed on the Ag nanoparticles through its chromophore group resulted in double amino-functionalized nanoparticles, while the amino functionals further targeted specific molecular species. Cresyl violet SERS-labeled nanoparticles described in Ref. [108] proved to be successful for use as SERS nanotags for interrogating skin tissue from ex vivo mice with induced melanoma [92]. The obtained results create future perspectives for progress in early cancer diagnostic based on amino-functionalized nanoparticles and SERS. The SERS fingerprints of tissues are sensitive to subtle chemical changes and can be used to track chemical modifications in tissues at the molecular level. SERS can be a valuable

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and powerful approach in early cancer diagnostic provided that reproducible output from a statistically relevant number of individuals is considered, in controlled experimental conditions.

CONCLUDING REMARKS Raman spectroscopy and its derived techniques can be successfully involved in various steps required for new drug discoveries utilizing natural sources. Raman spectroscopy techniques, such as molecular characterization, active ingredient identification, quantitative evaluation, molecular characterization of pharmaceutical formulations, understanding dependencies related to drug release and/or drug delivery, noninvasive molecular characterization of live tissue, monitoring pharmaceutical treatments and evaluating their effects, make it a great candidate for use in clinical trials and in making contributions to the yet unsolved problems in current medicine. Taking into account the rapid progress being made in health care, such as new drug discoveries, early diagnostic capabilities, the powerful approach of nanotechnology-based drug formulation, targeting, and theranostic approaches, we are confident that Raman spectroscopy will become an active player in the field of modern health care.

ABBREVIATIONS BA CD CRM DFT DMBA FIR FT GC–MS H&E HPLC IR MIR NE NIR NP SEM SERS TEM TPA UV

betulinic acid cyclodextrin confocal Raman microspectroscopy density functional theory 7,12 dimethylbenzanthracene far Infrared Fourier transform gas chromatography—mass spectrometry hematoxylin and eosin high-performance liquid chromatography infrared middle infrared nanoemulsion near-infrared nanoparticle scanning electron microscopy surface enhanced Raman spectroscopy transmission electron microscopy 12-O-tetradecanoylphorbol-13-acetate ultraviolet

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