Enhancing Extraction Yield and Purity of Mitragynine

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INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 03, MARCH 2020 ISSN 2277-8616 ENHANCING EXTRACTION YIELD AND PURITY OF MITRAGYNINE FROM MITRAGYNA SPECIOSA THROUGH SEQUENTIAL SOLVENT EXTRACTION AND CHARACTERISATION USING NMR TECHNIQUE Radhiahtul Raehan Mustafa, Rashidah Sukor, Siti Mariam Mohd Nor, Nazamid Saari and Farah Asilah Azri

Abstract—Mitragynine is the main alkaloid in Mitragyna speciosa which are commonly used to treat various illness. In this study, the extraction of mitragynine were performed using successive solvent extraction at different solvents‘ polarities. Purification of the extract produced 0.075 (g/g) of pure mitragynine. The Rf values was identified at 1.60 (hexane extract) and 0.80 (hexane-chloroform and hexane-chloroform-methanol extract). GC-MS analysis confirmed the presence of mitragynine in the extracts. Finally, 98% (w/w) of pure mitragynine was compared based on average intensity ratio of carbon signals to the standard. This study demonstrated high yield of pure mitragynine was successfully extracted from M. speciosa leaves. Index Terms— Column chromatography, GC-MS, mitragynine, NMR, polarity, solvent extraction and TLC ——————————  ——————————

1 INTRODUCTION PLANT herbs are the source of essential bioactive compounds weight los, muscle tremor and nystagmus [1] [2] [4] [5]. that are needed by humans for nutrients and supplements. Mitragynine or scientifically known as 9-methoxy-

Mitragyna speciosa is an ethnomedicinal plant herb that is corynantheidine, C23H30N24 (MW 398.50, Figure 1) is the main commonly found in the Southeast Asian region including active alkaloid in M. speciosa leaves, in which the content may Malaysia, Thailand, and Myanmar. In Thailand, this leaf is vary according to the geographical area. Due to its unique known as kratom, ithang, kakuam, kratom or thom, while in properties and potential application in drug discovery as well Malaysia, people call it biak-biak or ketum. Since the early as toxicology and pharmacological studies, it has attracted 19th century, the M. speciosa has been traditionally used as a many researchers to study their properties, extraction methods leaf poultice for treating muscle pain, diarrhea, cough, and and potential applications. For instance, several extraction intestinal infection [1] [2]. The leaves of M. speciosa can be techniques have been reported for the extraction of consumed in various ways including by boiling it in water for mitragynine from M. speciosa leaves such as the Soxhlet drinking, smoking it using bamboo pipe, and eating it fresh. extraction, [6], [7], [8], [9] methanolic and hot methanolic Previous studies have shown that drinking the juice of M. extraction, [10], [11]. [12] methanolic with pH modification speciosa leaves was common among drug users in the extraction, [13] ultrasonic-assisted extraction (UAE), [14] Northern States of Peninsular Malaysia to manage opiate ultrasonic-assisted, and freeze-dry extraction. [15] However, withdrawal symptoms after extended use [2]. The drug users the ultrasonic-assisted could cause the degradation of the commonly consume it in a form of cocktail with the addition of compound at high frequencies of ultrasound energy [16]. other ingredients including cola beverages, codeine- or Meanwhile, freeze-dry extraction method which is consider as diphenhydramine-containing cough syrup, mosquito coils, an expensive and complex method [17]. Besides, the Soxhlet herbicide, and many other toxic substances to enhance the extraction with methanol was found to be tedious and time- cocktail's effect. Besides Malaysia, M. speciosa is also consuming due to the slow process. In addition, the consumed in other countries including Sweden, Poland, subsequent purification method such as acidification or Denmark, Australia, Myanmar, Germany, United States, and neutralization process is needed to obtain pure mitragynine. the United Kingdom as traditional medicine for alcohol [6]. withdrawal and chronic pain [2] [3]. However, most countries have banned the usage of M. speciosa leaves due to addictive potential, toxicity and other health concerns such as nausea,

————————————————  Radhiahtul Raehan Mustafa (Mrs.), Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia  Rashidah Sukor (Dr.) (Corresponding author), Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia  Siti Mariam Mohd Nor (Dr.), Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia  Nazamid Saari, (Prof.) Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia  Farah Asilah Azri (Mrs.), Department of Food Science, Faculty of Food Science and Figure 1. Chemical structure of mitragynine Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor. 3846 IJSTR©2020 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 03, MARCH 2020 ISSN 2277-8616

A good extraction method especially for plant alkaloids is 2 MATERIALS AND METHODS vital to produce compounds with high purity. The purity of the 2.1 Chemical and solvents extract will be demonstrated by a well separated chromatographic peak for a targeted compound. It is also Organic solvents including n-hexane, chloroform, ethyl important to obtain pure compound from plant extract to acetate, and methanol used for preparing crude extracts were prevent undesirable side effects on the medical or of analytical grade and purchased from Fisher Scientific pharmacological application due to the change of chemical (Loughborough, UK). Pure mitragynine standard (98%) was properties of the compound. In order to obtain a pure purchased from Chromadex (Irvin, USA). Ultra-pure water compound, purification plays important role in the extraction (18.2 MΩ-cm) was obtained from the water purification system process. Basically, purification of target compound using silica (ELGA, Lane End, UK). gel by column chromatography is performed to separate the 2.2 Plant materials desired compound from mixture, based on their different absorption capabilities towards the silica gel. The M. speciosa leaves were obtained from Teluk Intan, Perak, effectiveness of column chromatography in isolating Malaysia. Authentication of the M. speciosa leaves was mitragynine [18], 7-hydroxyspeciociliatine [19], oxindole and verified by a botanist from Herbarium, Faculty of Forestry, indole alkaloid from M. speciosa leaves [20] were previously Universiti Putra Malaysia. The herbarium voucher specimens reported. A purified compound can be further analyzed using were deposited for future references (H 008). GC-MS [21], HPLC SFC [22], LC-MS [23] [24] [25] and UPLC- MS [26] to determine the efficiency of the extraction method. 2.3 Extraction of mitragynine Apart from obtaining a pure extract, the selection of a suitable Fresh leaves of M. speciosa (5.0 kg) were washed with water solvent for the extraction process is also crucial in producing a to remove dirt and air-dried at room temperature (28°C) for targeted compound with high purity [27]. The solvent selection seven days. The dried leaves were ground into fine powder is also driven by the characteristic of the targeted bioactive using a blender. The leaves were then soaked with solvents of compound (alkaloid) [28]. Solvents with different polarities different polarities, from non-polar (hexane), followed by have been widely used for isolating target compound from the intermediate polar (chloroform), and polar (methanol) mixture according to their relative polarities. Previous study according to previous study with some modification [29]. reported the use of solvents with different polarities in Briefly, M. speciosa leave powder (1.5 kg) was soaked in 2.5 L extracting antioxidant compounds including flavonoids, of hexane at room temperature to remove fat. Mitragynine lignans, and xanthone from ethnobotanical [29]. In another from defatted M. speciosa leaves were then sequentially study, similar use of solvents with different polarities was extracted using chloroform, followed by methanol under similar applied for extraction of major polyphenols in ―misai kucing” conditions. The extraction and evaporation process using (Orthosiphon stamineus), which showed significant free rotary evaporator were repeated three times. Fractions from radical-scavenging activity [30]. In sequential extraction the same solvent extracts were pooled, filtered using filter method, a non-polar solvent (e.g., hexane, petroleum ether) is paper (Whatman, 125 mm Ø) and further concentrated using a first used to extract the non-polar compounds such as fat, rotary evaporator (BUCHI, Flawil, Switzerland) at 40ºC. The wax, chlorophyll, pigments, alkane, fatty acid and sterols. The dried crude extracts from different solvents were subjected to intermediate polar solvents (e.g., chloroform, dichlorometane) TLC and GC/MS analyzes. The flow of the extraction process is then used to further solubilise the intermediate polar is shown in Figure 2. compounds such as flavonoids and lipophilic compounds. Finally, the high polar solvents such as methanol and ethanol 2.4 Determination of mitragynine content at each are commonly used to extract high polar compounds including extraction stage alkaloid, tannis and other hydrophilic compounds [31]. The 2.4.1 Thin layer chromatography (TLC) efficient extraction of mitragynine with a combination of polar The crude extracts from each extraction stage were monitored and non-polar organic solvents were previously reported [23] using a 6 cm × 4 cm silica gel 60 F254 sheets (0.040–0.063 [27]. Hence, our study employed a combination of polar and mm, Merck KGaA, Germany) as stationary phase. The spots non-polar organic solvents to extract mitragynine from M. were developed in hexane and ethyl acetate (3:2) and speciosa leaves.In this study, we report the extraction of the visualized under UV at 254 nm. Identification of UV inactive mitragynine from M. speciosa leaves using a sequential compound was carried out by staining with potassium solvent extraction method. The non-polar solvent of hexane permanganate (KMnO ) solution. The spots were observed on was first employed to remove wax, chlorophyll, and pigment. 4 Then, chloroform was used as the intermediate solvent while, the TLC plate and Rf values were recorded. methanol was employed as the high polar solvent to extract the mitragynine. This sequential extraction technique offers a 2.4.2 Gas Chromatography/Mass Spectrometry (GC/MS) The GC/MS analysis of mitragynine from different crude simpler method over other methods with high yield of extracts was performed using a gas chromatographic system mitragynine from M. speciosa leaves. The extracted and (GC-2010 Plus) coupled with GCMS-QP2010 Ultra Mass purified mitragynine can be further used for various Spectrometry Detector (Shimadzu, Tokyo, Japan) and applications, where standard is limited and extremely costly. separated on a BPX5 column (30.0 m length × 0.25 mm ID × 0.25 µm). Injection volume (1.0 µL) was performed in split flow control mode with the ratio of 1:10. Helium was used as carrier gas at a flow rate of ± 1 mL/min and the temperature of

3847 IJSTR©2020 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 03, MARCH 2020 ISSN 2277-8616 column and oven were maintained at 50°C and 250°C, The signals for ¹H NMR were analyzed with an appropriate respectively. The mass range was scanned from 40 to 670 abbreviation for multiplicities as a singlet (s), doublet (d), triplet amu. The compounds were identified by comparing their (t), and multiplet (m). The integration curve for each proton retention times to mitragynine standard at major ion of m/z reflected the abundance of individual protons. 398.

3 RESULTS AND DISCUSSION 3.1 Extraction of mitragynine

An efficient natural product extraction greatly depends on the

extracting solvent and the characteristics of the targeted compound. The use of extracting solvents is usually affected by their polarities [32]. It can be defined by the solubility of the compound by the sum of all the molecular properties [33]. Previous studies have shown that, the extraction of a bioactive compound was done successfully using solvent with different polarities by which the initial step employed the usage of a non-polar solvent i.e., hexane, followed by chloroform, ethyl acetate, and butanol [34] [35] [36]. The principle of ‗like dissolves like' was implemented in the extraction process where, the non-polar solvents will extract out the non-polar active compound and the polar compound will be extracted by polar solvents [37]. Thus, the M. speciosa leaves which consists of multi-bioactive alkaloids will be easily extracted according to their polarities. Mitragynine were previously extracted using single solvent of methanol [6] [15] [38] [39]. By employing methanol as one of the extracting solvents with combination of other solvents with varying polarities, 12.3 g/kg Figure 2. Schematic diagram of a successive mitragynine extraction using solvent with different polarities (i.e., hexane, of crude mitragynine extract was obtained from this study. The chloroform and methanol). selection of solvent used for extraction mostly depends on the solvent polarity. Based on the Snyder polarity index, the order 2.4.3 Purification of mitragynine using column of the polarities for three solvents used in our study were chromatography hexane (P′) (0.1) > chloroform (P′) (4.1) > methanol (P′) (5.1) From the TLC result, hexane-chloroform-methanol crude [40]. The solvent polarity was determined by the solubility of extract was further purified using column chromatography, the compound, which depends on the interaction forces packed with silica gel 60 (200-300 mesh, 40-63 mm, between the solvent and solute [33]. The sequential extraction Darmstadt, Merck, Germany). For column packing, the column method using solvent with different polarities used in our study was rinsed with acetone to remove dirt and dried. Then, glass produced a higher yield of mitragynine (75.0 mg) as compared wool was inserted at the bottom of the column and a slurry of to previous reports [6] [8] [10] [11] [19]. This could be silica gel (50 g) in hexane (55 mL) was poured into the glass contributed by the use of solvent combination such as hexane, column (40 x 300 mm). Sea sand (1 cm, 50–70 mm) was chloroform, and methanol during the extraction process that added at the bottom of the column to prevent sample may have reduced the loss of targeted compound and lead to distraction during addition of the solvent. Hexane was allowed higher chances of recovery of the mitragynine extract in the to flow during the packing process to the level of 1 cm above extract.The primary solvent, i.e., hexane is a non-polar the stationary phase. The packed column was kept overnight solvent, was primary used in our study for defatting purpose. It to remove bubbles. Crude extract (ca. 1 g) was ran through has been widely used in removing non-polar compounds such the packed column in hexane: ethyl acetate (3:2) as a mobile as plant pigments, waxes, resin, and chlorophyll [28]. The use phase at a flow rate of 3 mL/min. Approximately 15 mL of of hexane in the preliminary extraction and washing step was mobile phase was collected for each fraction and analyzed on employed from previous study for the extraction of mitragynine the TLC plate. Similar spots of the residue on the TLC plate [6]. In our study, the intermediate polar solvent i.e., chloroform were then combined and concentrated in the rotatory and the high polar solvent i.e., methanol were used to extract evaporator to obtain the final product. the intermediate and high polar compounds from the plant. Chloroform is miscible, conveniently volatile and unreactive, 2.4.4 Charaterization of purified mitragynine extract by thus making it suitable to intermediate polar compound extract nuclear magnetic resonance (NMR) such as mitragynine [14]. By increasing the solvent polarities The spectra of ¹H NMR and 13C NMR were obtained from and using methanol as the final extracting solvent, polar JEOL JMTC Spectrometer (Tokyo, Japan) at 500 MHz and compounds were extracted with enriched alkaloid content. The

125 MHz, respectively using deuterated chloroform (CDCl3). strong attraction between mitragynine, a polar compound, and Chemical shifts (δ) were recorded in parts per million (ppm). a polar solvent, such as methanol and chloroform are equated 3848 IJSTR©2020 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 03, MARCH 2020 ISSN 2277-8616 to a greater solubility as compared to the weak attraction solvents obtained from TLC using hexane and ethyl acetate (3:2) between mitragynine and hexane which lead to lesser as mobile phase Distance solubility. Previous study investigated the use of solvents with travelled R Sample/Extract f Spot colour different polarities in extracting bioactive compounds from by solute values pepper [41]. Petroleum ether was adopted in the study as the (cm) primary non-polar solvent for extracting lipids. In another Hexane 6.40 1.60 Greenish study, hexane, chloroform and methanol were employed in the Hexane-chloroform 3.20 0.80 Greenish brown extraction of chemical constituent in Cinnamommun Hexane-chloroform- 3.20 0.80 Greenish brown microphyllum [29]. methanol Mitragynine standard 3.30 0.82 Greenish brown 3.2 Determination of mitragynine content at each Rf values of chloroform and methanol were similar with mitragynine extraction stage standard 3.2.1 Thin layer chromatography (TLC) TLC: Thin layer chromatography TLC is a cost-effective, quick, simple and the most common Rf: Retention factor; calculated as distance travelled by solute / distance method used for identification of targeted compounds in the travelled by solvent (4.0 cm) natural products. It is a preliminary step to separate the targeted compounds from the crude extract. After the 3.2.2 Gas Chromatography/Mass Spectrometry (GC/MS) sequential extraction of mitragynine, the crude extracts from GC/MS is an analytical technique which used to separate hexane, hexane-chloroform and hexane-chloroform-methanol individual target compound from the chemical mixture. were examined on TLC for the presence of mitragynine. The Mitragynine from sequential extraction was further identified mitragynine content in each extraction stage was qualitatively using GC-MS. This chromatographic technique was able to identified with TLC using hexane: ethyl acetate (3:2) as the identify mitragynine from crude extract in each extraction stage mobile phase. The retention factor (Rf) of the crude extract in by separation process. The characteristic of the compound each stage were compared to the mitragynine standard. As can be identified in an optimized condition which produces a shown in Table 1, the Rf values of mitragynine in hexane- good separation of the compound from the mixture. The chloroform and hexane-chloroform-methanol extracts were attributes of mitragynine were determined using the GC-MS 0.8, which were comparable to the standard. This result is in analysis with a full scan mode (TIC) of 40 to 670 amu. The accordance with previous studies, who also found mitragynine spots at Rf comparable with the standard, however at different mitragynine peak in crude extracts was identified by value than we obtained in our study, i.e 0.54 and 0.92 comparing the retention time and mass spectrum to the respectively [38] [42]. The differences could be due to the mitragynine standard as shown in Figure 3.From the full scan dissimilarities in the mobile phase and ratio employed [43]. mass spectrum, the result indicates that mitragynine peaks Mitragynine, being polar was retained in the stationary phase, were identified in hexane-chloroform and hexane-chloroform- resulted in low Rf value of hexane-chloroform and hexane- methanol extracts at the retention time of 90 min. A strong ion chloroform-methanol extracts as compared to the hexane signal was obtained at m/z 398 was identified as the molecular extract. The Rf value of hexane extract was observed at 1.60 ion of mitragynine, which was in agreement with previous on TLC analysis. This indicates that, the extract contained reports [6] [14] [25]. Fragment ions at m/z 186, 214, 269, and non-polar compounds and suggests that mitragynine was 383 were similar with mitragynine standard. This observation absent in the extract. The non-polar compound in hexane also showed that the molecular ion was stable. No peak was extract was less retained on the stationary phase than the identified in hexane extract at 90 mins of retention time with polar compounds in hexane-chloroform and hexane- product ions of m/z 430 and 165, which confirms the absence chloroform-methanol extracts. From the TLC analysis, the of mitragynine in the extract. In addition, it was also observed variation in texture and colour of the solvent extracts were that two additional peaks appeared adjacent to the identified. The result demonstrates that, there were differences mitragynine at the retention time of 91 and 92 mins. Those in physical properties of crude extracts in each extraction peaks were identified as paynatheine and speciogynine, which stage. In crude hexane extract, the TLC analysis appeared as correspond to mitragynine analogues, with molecular masses a green-coloured spot which may be due to the presence of (M+) of m/z 396.20 and 398.50, respectively [15]. The GC-MS chlorophyll pigment [28]. In addition, the viscous characteristic results showed a satisfactory mitragynine extraction with of the extract is due to the presence of waxes and fats of the reduced impurities at each stage of the sequential solvent leaves [37]. Thus, the use of hexane is effective in removing extraction process. The major peak impurities at the retention waxes, chlorophyll pigments, and fat [44] which also correlates time of 75.0–80.0 from hexane extract to the hexane- with the finding in our study. However, the hexane-chloroform and hexane-chloroform-methanol extracts showed the chloroform-methanol extract were significantly reduced as greenish-brown spots with a slightly coarse texture, which solvent polarities increased. Result obtained from the GC-MS indicates the presence of mitragynine. The findings agreed analysis supports the TLC result and indicated that the with previous studies, who reported similar spot colour of mitragynine obtained from final crude extract after three greenish brown [6, 38]. solvent extractions had minimal impurities.

compounds were eluted earlier, followed by other compounds in its order of increasing polarity. This could be due to the properties of the silica gel adsorbent, the more polar compound such as mitragynine was strongly retained in the stationary phase, i.e. the silica gel [46].

3.2.4 Characterization of purified mitragynine extract by

nuclear magnetic resonance (NMR) The structure elucidation of the mitragynine was achieved by the NMR analysis in the 13C and ¹H spectra. The carbon and

proton number displayed the number of carbon and hydrogen

atom presented in the mitragynine with the chemical formula

of C H N O . NMR of 1H and 13C NMR spectra of purified 23 30 2 4 mitragynine were compared to the mitragynine standard which

was recorded in CDCl3 at room temperature. Chemical shifts (δ) are given relative to the mitragynine standard. Figure 4 shows the proton NMR spectra of purified mitragynine and standard. The integration shows that 30 hydrogen atoms were Figure 3. GC-MS chromatograms and mass spectra of similar to the mitragynine standard, which includes the number mitragynine standard (1A,1B) and different crude extracts: crude of hydrogen, nitrogen, and oxygen atom presented in the 1; hexane (2A,2B), crude 2; hexane, chloroform (3A,3B) and final mitragynine structure. crude; hexane, chloroform and methanol (4A,4B). Mass spectra of mitragynine are shown at peak retention time of 90 mins (pointed by arrow).

3.2.3 Purification of mitragynine using colum

The separation of mitragynine from the hexane-chloroform- methanol crude extract was performed using silica gel column chromatography. The solvent system used in column chromatography was similar with TLC, i.e., hexane: ethyl acetate (3:2). About 1.0 g of crude extract was purified by using column chromatography which produced 34 fractions of

15 mL mitrgaynine residues. The fractions attained were screened to determine the purity of mitragynine using TLC.

From the TLC observation, the 15th to 29th fractions had similar Rf values and spot characteristics of mitragynine as previously described. These fractions were then combined and concentrated using a rotary evaporator. The final product achieved from the column chromatography was approximately

75 mg of mitragynine (0.075 g/g). The mitragynine yield from our study was higher as compared to previous reports. Other Figure 4. 1H NMR spectra of (A) mitragynine standard and (B) studies have shown that mitragynine were produced at 0.0008 isolated mitragynine from final crude extract g/g using chloroform [6] and 0.03, 0.062 and 0.003 g/g using methanol extract [8] [11] [19]. Therefore, this study suggests The sharp signals at δ 3.6, δ 3.7, and δ 3.8 indicated the that the combination of solvent with different polarities H H H presence of methoxy groups at H-22, H-9, and H-17, which produces a higher yield of mitragynine as compared to the were comparable to the standard. The signal of methoxy extraction methods using single solvent. Table 2 summaries groups obtained were in agreement with the NMR the different extraction methods and yields of mitragynine spectroscopic data reported by previous study where, the obtained from previous studies in comparison with the present methoxy groups signals were found at 3.70 (3H, s, 22-OCH ), work. 3 3.87 (3H, s, 9-OCH ) and 3.71 (3H, s, 17-OCH ) [19]. Previous Table 2. Comparison of mitragynine yield between the present 3 3 study identified the signal peaks of methoxy groups at 3.84 work to other published methods (3H, s, 9-OCH ), 3.78 (3H, s, 17-OCH ) and 3.68 (3H, s, 22- In this study, the target compound, i.e. mitragynine, has a 3 3 polar characteristic which was eluted later from the column Extraction method Solvent used Yield References chromatography. The main factor that influences column (mg/g) chromatography performance was the molecular affinity Maceration Methanol 30.5 [8] Soaking Methanol 3.0 [19] between the solvent and solute, where the higher the polar Ultrasonication Methanol 62.0 [11] compound the more it will be retained in the column. Soxhlet Methanol 0.4 [10] Furthermore, the characteristics of the compound e.g., Soxhlet Petroleum ether; 0.88 [6] adsorption and solubility, influenced the adhesion strength to chloroform Sequential extraction Hexane, chloroform 75.0 Present work the absorbent [45]. Besides, other impurities and least polar and methanol 3850 IJSTR©2020 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 03, MARCH 2020 ISSN 2277-8616

OCH3) [6]. The presence of a single peak at downfield region 4 CONCLUSIONS at δ 7.9 ppm attributed to the N-H group. Similar signal of N- H Natural plant herb such as M. speciosa leaves consist of multi- H group was also reported in other studies at δ 7.8 (1H, br-s, H component mixtures of alkaloids. In summary, extraction of N-H) and δ 7.68 (1H, br-s, N-H) respectively [6] [19]. The H mitragynine by employing sequential extraction method and chemical shifts at δ 29.7, δ 40.6 and δ 111.4 were referred H H H supported by chromatographic techniques gave higher yield to as H-14, H-15, and H-16 which were comparable to the data and purity. Purification using silica gel column chromatography reported by previous finding and the mitragynine standard [8]. produced mitragynine (0.075 g/g) with a purity of 98% based Figure 5 shows a comparison of 13C NMR spectra of purified on average intensity ratio in NMR. This sequential extraction mitragynine with the mitragynine standard which represents 23 method offers an inexpensive technique with minimum carbons. All peaks identified were akin to the spectra of the technical knowledge and use of less complex technology. mitragynine standard. From the 13C spectra, three methoxy Therefore, the method is reliable and convenient to be used in groups were present with the signal obtained at δ 61.4, δ C C any laboratory with less tedious and minimise the need of 55.2, and δ 51.2, which were in agreement with the report by C using special instrument. Hence, pure mitragynine obtained previous study with similar groups reported at δ 61.6, δ 55.4 C C can be used for future application e.g., pharmaceutical, and δ 51.4 [6]. The chemical shift of C21 that represented C pharmacological and development of test kits. This study can C=O produced a signal at δ 69.21, which was comparable to C provide an additional knowledge to the present method of the the mitragynine standard (Table 3). All peaks were reflective of extraction target alkaloid in natural product with higher yield the number of actual mitragynine signals by comparing their and purity. NMR spectroscopic data with the previous reports [7] [8] [9]. The mitragynine obtained in this study had a purity of 98% based on the average intensity ratio between the carbon and ACKNOWLEDGMENT proton signals of mitragynine with those with a trace of This work was supported by the Ministry of Education, impurity observed at the baseline of the NMR [12]. A summary Malaysia under Grant [FRGS/UPM/5524655] and MyBrain of the carbon and proton NMR data analysis is shown in Table Scholarship. 3.The 1H and 13C NMR spectroscopic data of mitragynine obtained in this study were at par to the mitragynine standard and with those previously reported [6] [7] [8] [9] [19]. Hence, the present study shows a good yield of pure mitragynine was successfully isolated from M. speciosa leaves using sequential extraction method.

(A)

(B)

Figure 5. 13C NMR spectra of mitragynine standard (A) and isolated mitragynine from methanol extract (B).

Table 3. Analysis data of 13C NMR and 1H NMR for mitragynine

1H NMR δ 1H NMR δ* 13C NMR δ 13C NMR δ* Carbon/ (500 MHz, CDCl ) (500MHz, CDCl3, (125 MHz, CDCl3) (125 MHz, CDCl , Hydrogen 3 3 (isolated mitragynine) mitragynine standard) (isolated mitragynine) mitragynine standard)

N-H 7.93 (1H, br. s) 7.73 (1H, br. s) - - 2 - - 133.68 133.43 3.14 (1H, br. d, 3.11 (1H, br. d, 3 61.23 61.21 J= 10.3Hz) J= 10.3 Hz)

3.09-3.11 3.02-3.14 (2H, m) (2H, m) *5 53.68 53.67 2.43-2.50 2.45-2.52 (3H, m) (3H, m)

1.61 (1H, br. d, J=11.4 Hz) 1.62(1H, br. d, J=11.3 Hz) - - 3.09-3.11 3.02-3.14 *6 23.87 22.81 (2H, m) (2H, m) 7 - - 107.69 107.69 8 - - 117.53 117.53 9 - - 154.36 154.38 104.22 6.42 (1H, d, 6.42 (1H, d, 10 104.21 J=8.0 Hz) J=8.0 Hz)

6.97 (1H, dd, 6.97 (1H, d, 11 121.54 121.59 J=8.0 Hz) J=8.0 Hz) 6.88 (1H, dd, 6.88 (1H, d, 12 99.56 99.74 J=8.0 Hz) J=9.0 Hz) 13 - - 137.30 137.26

1.75-1.78 1.62-1.79 (2H, m) (2H, m) *14 29.77 29.79 2.43-2.50 2.45-2.52 (3H, m) (3H, m)

2.89-3.00 2.92-2.96 *15 40.61 40.62 (2H, m) (2H, m) 16 - - 111.45 111.43 17 7.41 (1H, s) 7.40 (1H, s) 160.44 160.42

18 0.84 (3H, d) 0.84 (3H, d) 12.79 12.79

1.18-1.19 1.20-1.25 (1H, m) (1H, m) *19 19.04 19.03 1.75-1.78 1.62-1.79 (2H, m) (2H, m)

1.61 (1H, d, 1.59 (1H, d, 20 39.87 39.82 J=11.4 Hz) J=11.4 Hz)

3.00-3.02 3.00-3.02 (2H, m) (2H, m) *21 57.69 57.59 2.43-2.50 2.45-2.52 (3H, m) (3H, m) 22 - - 169.22 169.25 22-COOCH3 3.69 (3H, s) 3.68 (3H, s) 51.22 51.27 9-OCH3 3.80 (3H, s,) 3.70 (3H, s,) 55.28 55.28 17-OCH3 3.85 (3H, s) 3.85 (3H, s) 61.45 61.48

a crude alkaloid extract derived from Mitragyna REFERENCES speciosa Korth. On permethrin elimination in rats,‖ [1] S. Ramanathan, S. Parthasarathy, V. Murugaiyah, E. Pharmaceutics. vol. 7, no. 2, pp. 10-26, 2015. Magosso, S. C. Tan, S. M. Mansor, ―Understanding [14] M. M. Sanagi, M. F. M. Ayob, N. Omar, W.A.W. the physicochemical properties of mitragynine, a Ibrahim, I Hussain, ―Determination of mitragynine for principal alkaloid of Mitragyna speciosa, for preclinical the identification of mitragyna species in Kedah evaluation,‖ Molecules. vol. 20, no. 3, pp. 4915–4927, (Malaysia) by gas chromatography-mass 2015. spectrometry,‖ Der Pharma Chem. vol. 5, no. 5, pp. [2] Z. Hassan, M. Muzaimi, V. Navaratnam, N. H. Yusoff, 131-138, 2013. F.W. Suhaimi, R. Vadivelu, B. K. Vicknasingam, D. [15] M.R.H.M. Haris, C. S. Lai, S. N. Idris, M. Abdul Amato, S. Von Horsten, N. I. Ismail, N. Jayabalan, A. Rahman, ―An optimized recovery of mitragynine from I. Hazim, S. M. Mansor and C.P. Müllerc, ―From Mitragyna speciosa using freeze drying and kratom to mitragynine and its derivatives: ultrasonic-assisted extraction method,‖ Int. J. Sci. physiological and behavioural effects related to use, Technol. vol. 15, no. 3, pp. 1077-1083, 2013. abuse, and addiction,‖ Neurosci. Biobehav, vol. 37, [16] N. Medina-Torres, T. Ayora-Talavera, H. Espinosa- no. 2, pp. 138-151, 2013. Andrews, ―Ultrasound assisted extraction for the [3] C. Veltri and O. Grundmann, ―Current perspectives on recovery of phenolic compounds from vegetable the impact of Kratom use,‖ Subst Abuse Rehabil. vol. sources,‖ Agronomy. vol. 47, no. 7, pp. 1-19, 2017. 10, pp. 23-31, 2012. [17] N. N. Azwanida, ―A review on the extraction methods [4] European Monitoring Centre for Drugs and Drug use in medicinal plants: Principle, strength and Addiction. ―The state of the drugs problem in Europe,‖ limitation,‖Med. Aromat. Plant. vol. 4, no. 3, pp. 3-8, Annual report 2010: EMCDDA. 2012. 2015. [5] R. V. Ancuceanu, M. Dinu, A. I. Anghel, O. C. [18] C.P. Kumar and J. Sachin, ―Extraction and isolation of Rebegea, O. T. Olaru, D. Popescu, G. Popescu, bioactive compounds from Ficus racemosa bark and ―Recent prohibition of certain psychoactive Cissampelos pareira root by chromatographic ―ethnobotanicals‖ in Romania,‖ Farmacia. vol. 58, no. techniques,‖ Int. J. Pharm. Sci. Rev. Res. vol. 20, no. 2, pp. 121-127, 2010. 2, pp. 101-106, 2013. [6] G. T. Beng, M. R. Hamdan, M. J. Siddiqui, M. N. [19] M. Kitajima, K. Misawa, N. Kogure, Y. Hatori, T. Mordi, S. M. Mansuri, ― A simple and cost effective Murayama, H. Takayama, I. M. Said, S. Horie, ―A new isolation and purification protocol of mitragynine from indole alkaloid, 7-hydroxyspeciociliatine, from the M. speciosa Korth (ketum) leaves,‖ Malaysian J. Anal. fruits of Malaysian Mitragyna speciosa and its opioid Sci. vol. 15, no. 1, pp. 54-60, 2011. agonistic activity,‖ J. Nat. Med. vol. 60, no. 1, pp. 28- [7] D. Ponglux, S. Wongseripipatana, H. Takayama, M. 35, 2005. Kikuchi, M. M. K. Kurihara, N. Aimi, D. Sakai, D, ―A [20] Z. Ali, H. Demiray, I. A. Khan, ―Isolation, new indole alkaloid, 7alpha- Hydroxy- 7H-mitragynine, characterization, and NMR spectroscopic data of from Mitragyna speciosa in Thailand,‖J. Nat Med. vol. indole and oxindole alkaloids from Mitragyna 60, no. 6, pp. 580-581, 1994. speciosa,‖ Tetrahedron Lett. vol. 55, no. 2, pp. 369- [8] P. J. Houghton, A. Latiff and I. M Said, ―Alkaloids from 372, 2014. Mitragyna speciosa,” Phytochem. vol. 30, no. 1, pp. [21] A. A. Philipp, M. R. Meyer; D. K. Wissenbach, A. A. 347-350, 1991. Weber, S. W. Zoerntlein, P. G. M. Zweipfenning, H. H. [9] K. L. R. Jansen and C. J Prast, ―Ethnopharmacology Maurer, ―Monitoring of kratom or krypton intake in of kratom and the mitragyna alkaloids,‖ urine using GC-MS in clinical and forensic toxicology,‖ Enthnopharmacol. vol. 23, no. 1, pp. 115-119, 1988. Anal. Bioanal. Chem. vol. 400, no. 1, pp. 127-135, [10] S. N. Harizal, S. M. Mansor, J. Hasnan, J. K. 2011. Tharakan, J. Abdullah,‖Acute toxicity study of the [22] M. Wang, E. J. Carrell, Z. Ali, B. Avula, C. Avonto, J. F. standardized methanolic extract of Mitragyna Parcher, I. A. Khan, ―Comparison of three speciosa Korth in rodent,‖ J. Enthnopharmacol. vol. chromatographic techniques for the detection of 131, no. 2, pp. 404-409, 2010. mitragynine and other indole and oxindole alkaloids in [11] R. Kikura-Hanajiri, M. Kawamura, T. Maruyama, M. Mitragyna speciosa (kratom) plants,‖ J. Sep. Sci. vol. Kitajima, H. Takayama, Y. Goda, ―Simultaneous 37, no. 12, pp. 1411-1418, 2014. analysis of mitragynine, 7-hydroxymitragynine, and [23] K. B. Chan, C. Pakiam, R. A. B. Rahim, ―Psycoactive other alkaloids in the psychotropic plant ―kratom‖ plant abuse: The identification of mitragynine in ketum (Mitragyna speciosa) by LC-ESI-MS,‖ Forensic and in ketum preparations,‖ Narcotics. vol. 57, no. 2, Toxicol. vol. 27, no. 2, pp. 67-74, 2009. pp. 249-256, 2005. [12] B. Janchawee, N. Keawpradub, S. Chittrakarn, S. [24] A. A. Philipp, D. K. Wissenbach, A. Weber, J. Zapp, S. Prasettho, P. Wararatananurak, K. Sawangjareon, ―A W. Zoerntlein, K. Kanogsunthornrat, H. H. Maurer, high-performance liquid chromatographic method for ―Use of liquid chromatography coupled to low- and determination of mitragynine in serum and its high-resolution linear ion trap mass spectrometry for application to a pharmacokinetic study in rats,‖ studying the metabolism of paynantheine, an alkaloid Biomed Chromatogr. vol. 21, no. 2, pp. 176-183, of the herbal drug kratom in rat and human urine,‖ 2007. Anal. Bioanal. Chem. vol. 396, no. 7, pp. 2379-2391, [13] K. Srichana, B. Janchawee, S. Prutipanlai, P. 2010. Raungrut, N. Keawpradub, ―Effects of mitragynine and [25] S. Lu, B. N. Tran, J. L. Nelsen, K. M. Aldous,

―Quantitative analysis of mitragynine in human urine extracts,‖ Int. J. Appl. Nat. Sci. vol. 1, no. 1, pp. 8-26, by high performance liquid chromatography-tandem 2012. mass spectrometry,‖ J Chromatogr. B: Anal. Technol. [38] D. Singh, S. Narayanan, C.P Muller, M. T. Swogger, Biomed. Life Sci. vol. 877, no. 24, pp. 2499-2505, N. J. Y. Chear, E. B. Dzulkapli, N. S. M. Yusoff, D. 2009. Ramachandram, F. Leon, C. R. McCurdy and B. [26] P. K. Vuppala, S. Jamalapuram, E. B. Furr, C. R. Vicknasingham. ―Motives for using kratom (Mitragyna McCurdy, ―Development and validation of a speciosa Korth.) among regular users in Malaysia,‖ J. UPLC‐MS/MS method for the determination of Ethnopharmacol. vol. 233, pp. 34-41, 2019. 7‐hydroxymitragynine, a μ‐opioid agonist, in rat [39] K. J. Sufka, M. J. Loria, K. Lewellyn, J. K. Zjawiony, Z. plasma and its application to a pharmacokinetic study. Ali, N. Abe, I. A. Khan, ― The effect of Salvia divinorum Biomed. Chromatogr. vol. 27, no. 12, pp. 1726-1732, and Mitragyna speciosa extracts, fraction and major 2013. constituents on place aversion and place preference [27] H. Takayama and T. Maruyama, ―Simultaneous in rats,‖ J. Ethnopharmacol. vol. 151, pp. 361-364, analysis of mitragynine, 7-hydroxymitragynine, and 2014. other alkaloids in the psychotropic plant "kratom" [40] J. J. Kirkland, ―Analytical Chemistry,‖ Journal of (Mitragyna speciosa) by LC-ESI-MS,‖ Forensic Chromatography Library, L. R Synder and J. J. Toxicol. vol. 27, pp. 67-74, 2009. Kirkland. New York: John Wiley & Sons. pp 431-482, [28] S. Sasidharan, Y. Chen, D. Saravanan, K. M. 1979 Sundram, Y. L. Latha, ―Simultaneous analysis of [41] C. Kouassi, ―Profiles of bioactive compounds of some mitragynine, 7-hydroxymitragynine, and other pepper fruit (Capsicum L.) varieties grown in Côte alkaloids in the psychotropic plant "kratom" (Mitragyna d‘Ivoire, ―Innov. vol. 11, no. 9, pp. 23-31, 2012. speciosa) by LC-ESI-MS,‖ Afr. J. of Tradit. [42] S. Parthasarathy, J. B. Azizi, S. Ramanathan, S. Complement and Altern. Med. vol. 8, no. 1, pp. 1-10, Ismail, S. Sasidharan, M. I. M. Said, S. M. Mansor, ― 2011. Evaluation of antioxidant and antibacterial activities of [29] M. A. Norazah, M. Rahmani, S. Khozirah. H. B. M. aqueous, methanolic and alkaloid extracts from Ismail, M. A. Sukari, A. M. Ali, G.C.L Ee, ―Chemical Mitragyna speciosa (rubiaceae family) leaves,‖ constituents and antioxidant activity of Cinnamomum Molecules. vol. 14, pp. 3964-3974, 2009. microphyllum,” Pertanika J. Sci. Technol. vol. 18, no. [43] M. Kotzé, J. N. Eloff, P. J. Houghton, “Extraction of 2, pp. 263-270, 2010. antibacterial compounds from Combretum [30] G. A. Akowuah, Z. Ismail, I. Norhayati, A. Sadikun, microphyllum (Combretaceae),‖ S. Afr. J. Bot. vol. 68, ―The effects of different extraction solvents of varying no. 1, pp. 62-67, 2016. polarities on polyphenols of Orthosiphon stamineus [44] P. Cos, A. J. Vlietinck, D. V. Berghe, L. Maes, ―Anti- and evaluation of the free radical-scavenging activity,‖ infective potential of natural products: How to develop Food Chem. vol. 93, no. 2, pp. 311-317, 2005. a stronger in vitro ‗proof-of-concept‘,‖ J. [31] V. Seidel, ―Initial and Bulk Extraction of Natural Ethnopharmacol. vol. 106, no. 3, pp. 290-302, 2006. Products Isolation,‖ Springer Nature- Methods in [45] B. A. Olsen, ―Hydrophilic interaction chromatography Molecular Biology, S. D. Sarker and L. Nahar, eds., using amino and silica columns for the determination Natural Products Isolation, Springer Science and of polar pharmaceuticals and impurities, ―Afr. J. Food Business Media, pp. 27-41, 2012. Sci. vol. 913, no. 1, pp. 113-122, 2001. [32] M. Romdhane and C. Gourdou, ―Investigation in solid- [46] S. Bawazeer, O. B. Sutcliffe, M. R. Euerby, ―A liquid extraction: influence of ultrasound,‖ Chem. Eng. comparison of the chromatographic properties of silica J. vol. 87, no. 1, pp. 11-19, 2002. gel and silicon hydride modified silica gels,‖ Afr. J. [33] F. Lin and M. M, Giusti, ―Effects of solvent polarity and Food Sci. vol. 1263, no. 9, pp. 61-67, 2012. acidity on the extraction efficiency of isoflavones from soybeans (Glycine max),” J. Agric. Food Chem. vol. 53, no. 10, pp. 3795-3800, 2005. [34] I. Ahmad, Z. Anwar, K. Qadeer, A. Sabah, A. Arif, A. Arsalan, ―Effect of solvent polarity on the extraction of components of pharmaceutical plastic containers,‖ Pak. J. Pharm. Sci. vol. 30, no. 1, pp. 247-252, 2017. [35] M. A. Hossain, S. S. Al-Hdhrami, A. M. Weli, Q. Al- Riyami, J. N. Al-Sabahi,‖ Isolation, fractionation and identification of chemical constituents from the leaves crude extracts of Mentha piperita L. grown in Sultanate of Oman,” Asian Pac. J. Trop. Biomed. vol. 4, no. 1, pp. 368-372, 2014. [36] T. U. Nwabueze and K. S. Okocha, ―Extraction performances of polar and non-polar solvents on the physical and chemical indices of African breadfruit (Treculia africana) seed oil,‖ Afr. J. Food Sci. vol. 2, no. 2, pp. 119-125, 2008. [37] A. Gupta, M. Naraniwal and V. Kothari, ―Modern extraction methods for preparation of bioactive plant