E m T N A N a b c d e f a A R R A A K M T Q I A A 1 g c t w g c N h 0 Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 475–480 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur na l ho mepage: www.elsev ier .com/ locate / jpba stablishment of a quantitative and qualitative analysis and isolation ethod for tetracyclic iridoids from Morinda lucida Bentham leaves omoe a Ohta , Tanatorn Tilkanontb, Frederick Ayerteyc, Mina Nakagawaa, guyen Huu Tunga,d , Peter Bolahc, Heron Blagogee Jnr. c, Alfred Ampomah Appiahc, ugustine Oclooc, Mitsuko Ohashie,f, Kensuke Tanouea, Yasuchika Yamaguchia , obuo Ohtaf, Shoji Yamaokaf, Shiro Iwanagaf, a Takuhiro Uto , Yukihiro Shoyamaa,∗ Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Nagasaki 859-3298, Japan Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand Centre for Plant Medicine Research, P. O. Box 73, Mampong, Akuapem, Ghana School of Medicine and Pharmacy, Vietnam National University, Hanoi, Viet Nam Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, P. O. Box LG 581, Legon, Ghana Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan r t i c l e i n f o a b s t r a c t rticle history: A new high performance liquid chromatography (HPLC) method has been established for quantitative eceived 21 September 2018 and qualitative analysis of three tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3), which are eceived in revised form 25 October 2018 responsible for anti-trypanosomal and anti-leishmanial activities of Morinda lucida Bentham leaves. Sep- ccepted 25 October 2018 aration of 1–3 from dried 80% aqueous (aq.) ethanol extract was achieved on a reversed-phase cholester vailable online 29 October 2018 column packed with cholesteryl-bonded silica using an acetonitrile-0.1% aq. formic acid mobile phase system. Ultraviolet-visible (UV-VIS) spectroscopy was employed for detection of compounds, and their eywords: contents were determined by measuring absorbance at 254 nm. Depending on the above system, several orinda lucida etracyclic iridoids factors potentially affecting the concentration of tetracyclic iridoids were evaluated resulting in several uantitative analysis variation on plant organs, seasonality, variation between individual trees, and branch positions within solation the trees. Moreover, we developed a simple, quick, and effective method for tetracyclic iridoid isolation nti-trypanosoma from M. lucida leaves that consisted of extraction by sonication into 80% aq. ethanol, basic hydroly- nti-leishmania sis, acid neutralization, liquid-liquid extraction into an organic solvent, and reverse phase open column chromatography. Employing this method, we have succeeded to obtain 1 as a colorless crystal yielding of 0.23%, which was 28 times higher than that of previous isolation method. Setting up methodology in this paper may be important for future in vitro and in vivo studies of tetracyclic iridoids and moreover for their applications in new drug design and development. © 2018 Elsevier B.V. All rights reserved.. Introduction Morinda lucida Bentham (Rubiaceae), a medium-sized ever- reen tree with dark green, and shiny leaves, is widely grown and ultivated in West and Central Africa [1]. It has been used as a tradi- ional remedy for fever, dysentery, abdominal colic, and intestinal orm infestation [2,3]. M. lucida extracts have been investi- ated frequently resulting in several activities like anti-oxidant, ∗ Corresponding author at: Department of Pharmacognosy, Faculty of Pharma- eutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, agasaki 859-3298, Japan. E-mail address: shoyama@niu.ac.jp (Y. Shoyama). ttps://doi.org/10.1016/j.jpba.2018.10.044 731-7085/© 2018 Elsevier B.V. All rights reserved.anti-diabetic, anti-cancer, anti-bacterial, anti-trypanosomal, and anti-malarial activities [4–9]. Phytochemical studies of M. lucida have shown that it contains alkaloids, anthraquinones, triterpenes, and iridoids [1,10–12]. Previously, we found that the extract of M. lucida leaves had strong activity of anti-tripanosomal parasites [13]. In addition, we isolated and elucidated chemical structures of three novel tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3) (Fig. 1) from M. lucida leaves [14–16]. The absolute configuration of 2 was determined by using X-ray analysis and confirmed as an enantiomer of oruwacin [14]. Moreover, we confirmed that 1–3 exhibited strong anti-trypanosomal, anti-leishmanial, and anti- malarial activities in vitro and in vivo. [16,17]. In trypanosomes, 1 and 3 induced significant apoptosis-like cell death [15,16]. It is sug- gested that tetracyclic iridoids hence might be the potential lead or 476 T. Ohta et al. / Journal of Pharmaceutical and B s o l e y o M s t e i 2 2 p ( t p c i i ( f T 6 v i 2 f p ( S i o T t i b G t t e f 2 a Fig. 1. Chemical structures of 1–3. eed compounds of anti-trypanosomal agents. However, the yields f 1–3 from dried leaves were extremely low in our previous study ike 0.0045%, 0.0032%, and 0.0005%, respectively [15,16]. Herein, we stablished a new method for the quantitative and qualitative anal- sis of 1–3 to find superior plants containing higher concentration f tetracyclic iridoids. We then examined the quantities of 1–3 in . lucida leaves, stems, and roots, as well as potential influences of easonality, variations between individual trees, and branch posi- ions on the contents of the compounds. Furthermore, we report an fficient, rapid, and facile isolation and purification of tetracyclic ridoids. . Materials and methods .1. General procedures Specific rotations were measured with a DIP-360 digital olarimeter (JASCO, Easton, USA). Nuclear magnetic resonance NMR) spectra were recorded on a JEOL ECX 400 FT-NMR spec- rometer (JEOL, Tokyo, Japan) at 20 ◦C using JEOL’s standard pulse rogram, with tetramethylsilane as the internal standard and hemical shift values were expressed in ı (ppm). Electrospray onization time-of-flight mass spectrometer (ESI-TOF-MS) exper- ments employed a Waters Xevo G2-XS Q-TOF mass spectrometer Waters, Milford, MA, USA). Column chromatography was per- ormed on YMC ODS-A gel (50 m, YMC Co. Ltd., Kyoto, Japan). hin-layer chromatography (TLC) was performed on TLC Silica gel 0 RP-18 F254S (Merck, Damstadt, Germany) plates. Spots were isualized by spraying with 10% aq. sulfuric acid, followed by heat- ng. .2. Plant materials All plant samples were derived from M. lucida. ML-S was made rom leaves collected in Mampong, Ghana on January 2016. Sam- les used to analyze contents of 1–3 in different plant parts leaves, stems, and roots) were collected in Ayikuma, Ghana on eptember 2017. To evaluate the potential impact of seasonal- ty on the amounts of 1–3, twelve leaf samples were collected ver a period of March 2017–February 2018 in Ayikuma, Ghana. o assess the variability of compound contents among different rees, and leaf samples were collected from twenty individual trees n Ayikuma, Ghana. Leaf samples used to evaluate the impact of ranch positions were collected from tree 6 and 16 in Ayikuma, hana on December 2017. The samples were identified by one of he author (Y. S.) and voucher specimens have been deposited at he Department of Pharmacognosy, Faculty of Pharmaceutical Sci- nces, Nagasaki International University, Japan and at the Centre or Plant Medicine Research, Ghana..3. Reagents and chemicals Ethanol (EtOH), methanol (MeOH), ethyl acetate (EtOAc), and cetonitrile (MeCN) were purchased from Nacalai tesque Inc.,iomedical Analysis 164 (2019) 475–480 Kyoto, Japan. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) which were used in isolation, purchased from Wako Pure Chemical Industries, Ltd., Japan. The reference samples of 1–3 were isolated from M. lucida leaves [15,16]. The NMR and HPLC of each reference samples showed high homogeneity, and the purity was accessed by HPLC using an UV-VIS detector to be more than 95%. Other chemicals than above were purchased from Wako Pure Chemical Industries, Ltd. 2.4. Preparation of sample solution For each sample, 20 mg of pulverized powder was suspended in 1 ml of either MeOH, EtOH, 80% aq. EtOH, 50% aq. EtOH, or MeCN. Extraction was performed by sonication at 40 kHz for 15 min. Extracts were then centrifuged at 12,000 rpm for 10 min. Each supernatant was then filtered with a filter vial (Standard Filter Vial PVDF 0.45 m, Thomson Instrument Co., Oceanside, USA), and a 10 l aliquot of each filtrate was analyzed by HPLC. 2.5. Preparation of standard solution A stock standard solution was prepared by placing 1 (4.00 mg), 2 (3.00 mg), and 3 (2.00 mg) in a 10 ml volumeric flask and adding 80% aq. EtOH to the mark. The solution contained 1, 2, and 3 at concentrations of 400 g/ml, 300 g/ml, and 200 g/ml, respec- tively. To prepare working solutions, aliquots of 8, 40, 200, and 1000 l of the stock standard solution were transferred to 5 ml volumeric flasks and diluted to the mark with 80% aq. EtOH. The working solutions were used to construct calibration curves. (1: 80 g/ml, 16 g/ml, 3.2 g/ml, 0.64 g/ml; 2: 60 g/ml, 12 g/ml, 2.4 g/ml, 0.48 g/ml; 3: 40 g/ml, 8 g/ml, 1.6 g/ml, and 0.32 g/ml). Calibration curves were made by injecting a 10 l aliquot of each working solution into the HPLC sys- tem. 2.6. HPLC instruments and conditions for 1–3 The HPLC system was performed on a JASCO LC-2000 Plus series (JASCO, Tokyo, Japan) equipped with an intelligent UV-VIS detec- tor (UV-2075 Plus), a quaternary gradient pump (PV-2089 Plus), an intelligent column oven (CO-2065 Plus), an intelligent autosampler (AS-2057 Plus), an interface (LC-Net II/ADC), and a data software (ChromNAV). A COSMOSIL Cholester column (4.6 × 250 mm, par- ticle size 5 m, Nacalai Tesque INC. Kyoto, Japan) was used for the HPLC analysis ◦ at 40 C with mobile phases MeCN and 0.1% aq. formic acid eluted according to the following gradient pro- gram: 0–12 min (32:68, v/v) → 12–15 min (32:68 → 50:50, v/v) → 15–28 min (50:50, v/v, hold). The flow rate was 1 ml/min, the injection volume was 10.0 l, and the detection wavelength was 254 nm. Peaks for 1, 2, and 3 were observed at 14.5 min, 21.4 min, and 24.8 min, respectively. 2.7. Calibration, validation, and application of established protocol Calibration curves were constructed with five standards pre- pared over the following concentration ranges: 0.64–400 g/ml (1), 0.48–300 g/ml (2), and 0.32–200 g/ml (3). Fresh calibration curves were prepared each day. Calibration curves were con- structed by plotting concentration (g/ml) on the horizontal axis and peak area (V*sec) on the vertical axis. Linearity was deter- mined using correlation coefficient (r2). ML-S extracts served as matrix standards and were used to determine the precision and accuracy of the analytical method. Intra-day precision was deter- mined by performing five injections from the same standard within and Biomedical Analysis 164 (2019) 475–480 477 a d R ( A c 2 w o i w w fi 0 t t t T c M a i w 3 3 e ( ( t o s s F T. Ohta et al. / Journal of Pharmaceutical day. Inter-day precision was determined with injections of stan- ards at the same concentration made over the course of five days. ecovery was determined by standard addition, in which 15 g/ml 1), 5 g/ml (2), and 3 g/ml (3) were added to sample solutions. content (%, w/w) was calculated as (contained amount of the ompound/dried material weight) × 100. .8. Extraction and isolation Dried and pulverized ML-S (350.66 g) was extracted six times ith 1.50 l volumes of 80% aq. EtOH by sonication at 40◦ C for ne hour. The extracts were combined and evaporated to dryness n vacuo. After removal of solvents, fractionation was performed ith the obtained residue (109.47 g). One of the extracts (107.77 g) as suspended in 4% aq. NaOH (1 l) and partitioned into 1 l of EtOAc ve times. The EtOAc fraction was then washed three times with .20 l of 4% aq. NaOH. The water fractions were combined and neu- ralized with 6% aq. HCl (0.90 l). Neutralized fraction was extracted hree times with 1 l of EtOAc per extraction. The EtOAc fraction was hen washed three times with water (1 l per wash) and evaporated. he EtOAc fraction (13.40 g) was analyzed on a reversed-phase ODS olumn ( 70 mm × 200 mm) using isocratic elution with 38% aq. eOH (22 l). The EtOAc fraction was found to contain 1 (981.2 mg) t a yield of 0.23%. Identity of 1 was confirmed by comparison of ts physical data ([˛] , 1H-NMR, 13D C-NMR, and mass spectrometry) ith reported values [14–16]. . Results .1. Optimal solvent system for preparation of sample solution Frist, the extraction conditions were examined to optimize the xtraction efficiency for the concentration of the target compounds 1–3). The contents were compared using five solvent systems MeOH, EtOH, 80% aq. EtOH, 50% aq. EtOH, or MeCN) under sonica- ion for 15 min. As shown in Fig. 2, 80% aq. EtOH (0.77%, contents f total tetracyclic iridoids) showed most efficacy than the other olvent systems resulted that 80% aq. EtOH was employed in this tudy. ig. 3. Typical HPLC chromatograms of a standard solution mixture (1: 400 g/ml, 2: 300Fig. 2. Contents of 1–3 in solvent systems (MeOH, EtOH, 80% aq. EtOH, 50% aq. EtOH, and MeCN). 3.2. Optimization of HPLC conditions Following the sample preparation method, the 80% aq. EtOH extract was analyzed the determination of 1–3 concentration by using HPLC. Typical HPLC chromatograms for a standard mixture of 1–3 and the ML-S extract are shown in Fig. 3. Baseline separation of peaks corresponding to 1–3 was achieved. Retention times of 1, 2, and 3 were 14.5 min, 21.4 min, and 24.8 min, respectively. Peaks in the extract were identified by the comparison of their retention times with those of the standards. 3.3. Validation of the assay system Linearity, intra- and inter-day precision, and accuracy were evaluated as shown in Table 1. Linear regression equations for the calibration curves were y = 14501x + 5350 (1), y = 16794x + 3533 (2), and y = 15893x + 636 (3), where x was the concentration (g/ml) and y was the peak area of the analyte (V*sec). Each of calibration curves was linear 2 in the range studied with r of 0.999. Intra-day relative standard deviation (RSD) values ranged from 0.72 to 2.41%, while inter-day RSD values ranged from 2.93 to 3.85%. Accuracy was g/ml, 3: 200 g/ml) (A) and an 80% aq. EtOH extract of ML-S (20 mg/ml) (B). 478 T. Ohta et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 475–480 Fig. 4. Contents of 1–3 in the extracts of plant organs (A), months (B), individual trees (C), different sampling branch positions (D2), and the detail of branch positions collected from tree 6 (D1). Table 1 Linearities, range, precisions, and recoveries for 1–3 from M. lucida leaves. Precisionb (RSD, %) Analyte Regression Equationa Correlation Coefficient Range c,d (g/ml) Recovery (RSD, %) Intraday Interday 1 y = 14501x + 5350 0.9999 0.64–400 2.41 2.93 103.67 ± 2.86 2 y = 16794x + 3533 0.9999 0.48–300 1.09 3.58 96.05 ± 1.37 3 y = 15893x + 636 0.9999 0.32–200 0.72 3.85 95.57 ± 2.73 a In the regression equation, x is the concentration of the analyte solution (g/ml), and y is the peak area of the analyte (V*sec). b Precision of the analytical method were tested using 80% aq. EtOH extracts of ML-S (n = 5). c Recoveries spiked with 80% aq. EtOH extracts of ML-S. d Values are means ± RSD (n = 5). T. Ohta et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 475–480 479 olatio d l o o 3 d w c i w i 0 p m o c i t 1 C 1 1 t S o c t w i p c T t i Fig. 5. Comparison of a developed is etermined through recovery experiments using extract from M. ucida leaves. Recovery rates were 95.57–103.67% with RSD values f lower than 2.86%. The analysis method achieved a high degree f reproducibility and accuracy. .4. Application of established protocol The contents of 1–3 in leaves, stems, and roots of M. lucida were etermined using the validated protocol. Fig. 4A shows that 1–3 ere not detected in the extracts of stems and roots. Only the leaves ontained tetracyclic iridoids (0.053%, contents of total tetracyclic ridoids). Twelve samples of M. lucida leaves collected in different months ere analyzed. Results are shown in Fig. 4B. Contents of 1–3 n extracts from April, May, and June were 0.063%, 0.060%, and .061%, respectively. The extracts of April, May, and June sam- les were richer in the target compounds than extracts from other onths. Tetracyclic iridoids were scarcely detected in the extracts f October and November samples. The concentration of target ompounds in the samples varied monthly and 3 was not found n all samples. Twenty samples collected from different trees were analyzed for heir 1–3 contents. As shown in Fig. 4C, extracts from trees 6 and 6 were especially rich in tetracyclic iridoids, due to high 2 content. onversely, 1–3 were not found in extracts from trees 7, 15, 18, and 9. Samples collected from different branch positions on trees 6 and 6 were also analyzed. Ten samples were collected from individual rees. Details of sample positions in the tree 6 are shown in Fig. 4D1. amples 6A-1∼6A-5 were collected from the individual positions f bottom branch, and samples 6B-1∼6B-5 were collected from the enter position of individual branch. Samples from tree 16 (16A-1 o 16A-5 and 16B-1 to 16B-5) were collected by the same manner ith the samples from tree 6. As shown in Fig. 4D2, the tetracyclic ridoid contents in samples 16A-1∼16A-5 tended to increase with roximity to the joint of the branch, while the tetracyclic iridoid ontents in the position 4 was the highest in samples 6A-1∼6A-5. he tetracyclic iridoid contents in sample group B increased until he position 4 and then decreased. Bigger differences in tetracyclic ridoid contents were observed in group B than that of group A.n method (A) and previous one (B). 3.5. An efficient, quick, and easy system for tetracyclic iridoids isolation Next, we investigated an efficient, quick, simple and higher yielding isolation method for tetracyclic iridoids. Since the isolation process consisted of complicated pathway in our previous method as indicated in Fig. 5B , we changed and simplified three points of a method. First, we incorporated 80% aq. EtOH as the extraction solvent because the concentration of tetracyclic iridoids was much higher than that of 50% aq. EtOH as indicated in Fig. 2 resulting in the increase of tetracyclic iridoid contents from 0.63% to 0.77%. Next, we tried to isolate all tetracyclic iridoids in the same manner with 1 for efficient purification. We found that 1 can be synthesized easily into 2 and 3. Since the carboxyl group at the C-4 position of 1 is acidic, it is possible to employ acid-base sep- aration. The extract was added to 4% aq. NaOH and fractionated with EtOAc. During the treatment, alkaline hydrolysis occurred to yield 1. The water fraction was then neutralized with 6% aq. HCl and fractionated with EtOAc to separate 1. Since it is known well that silica gel previously used formed a hydrogen bonding with carboxylic group of 1 easily, ODS column chromatography was employed for the isolation method. 1 eluting with 38% aq. MeOH. The newly developed isolation method of 1 was shown in Fig. 5A. With this new method, the yield of 1 increased from 0.0083% to 0.23%. 4. Discussion We developed an HPLC method for quantitative and qualita- tive analysis of three tetracyclic iridoids 1–3 from M. lucida. To our knowledge, this is the first report describing the quantitative and qualitative analysis of these compounds. Using this method, we examined the quantities of 1–3 in different plant organs, as well as other factors that could affect tetracyclic iridoid contents in M. lucida. The seasonal variation of secondary metabolite and differ- ences depending on the place of plant organs were well known. Previously we solved the seasonal variation against aconitine-type alkaloids contained in bulb using the clonally propagated Aconi- tum carmichaelii plants resulting that the aconitine-type alkaloids were accumulated approximately 3 times in early spring compared 4 and B t t s F i d s t a M p [ T i [ w c [ l i s [ p i f o p d [ i C [ A P J [ e G i J R [ [ [ beween forskolin content and growth environments in clonally propagated Coleus forskohlii, Biotronics 24 (1995) 1–6.80 T. Ohta et al. / Journal of Pharmaceutical o that of autumn [18]. Also we made clear that the concentra- ion of forskolin was different between the position of organs like tem and tuber using anti-forskolin monoclonal antibody [19,20]. rom these evidence, we set up seasonal variation, variations in ndividual trees, and sampling branch positions. It is notably evi- ent that only leaves contained tetracyclic iridoids. With regard to easonality, the extracts of April, May, and June leaf samples con- ained higher contents of the compounds. Ghana has two seasons, dry season from November to February and a rainy season from arch to October. Based on our results, we concluded that sam- le collection would best be performed during the rainy season. rees 6 and 16 had the highest concentration of tetracyclic iridoids n our analysis of the individual trees. When different positions ithin individual trees (6 and 16) were evaluated for their tetra- yclic iridoid contents, contents were higher near the knags and ower branches. This indicates old leaves accumulated tetracyclic ridoids. These phenomena could be useful for selecting M. lucida amples containing highest concentration of 1–3. We also evaluated an efficient, quick, and simple method for urification and isolation of tetracyclic iridoids. With our newly solation method, the yield of 1 increased 28-fold compared to our ormer method. The improved separation system yielded a col- rless, purified crystalline form of 1 quickly. Herein, it became ossible to isolate purified tetracyclic iridoids easily and abun- antly. These findings will facilitate investigation of tetracyclic ridoids as anti-trypanosomal agents. onflict of interest None. cknowledgements This research is supported by Science and Technology Research artnership for Sustainable Development (SATREPS) grant from the apan Science and Agency (JST) and the Japan International Coop- ration Agency (JICA) (2010 to 2015) and the Japan Initiative for lobal Research Network on Infection Diseases (J-GRID) from Min- stry of Education, Culture, Sports, Science & Technology (MEXT) in apan (2015 to present). eferences [1] H.O. Lawal, S.O. Etatuvie, A.B. Fawehinmi, Ethnomedicinal and pharmacological properties of Morinda lucida, J. Nat. Prod. 5 (2012) 93–99. [2] A.A. Odutuga, J.O. Dairo, J.O. Minar, F.A. 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