Selecting a green strategy on extraction of birch bark and isolation of

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Selecting a green strategy on extraction of birch bark and isolation of pure betulin using monoterpenes. Alexandr E. Grazhdannikov, Lyubov M. Kornaukhova, Vladimir I. Rodionov, Natalia A. Pankrushina, Elvira E. Shults, Anne Sylvie Fabiano-Tixier, Sergey A. Popov, and Farid Chemat ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00086 • Publication Date (Web): 06 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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ACS Sustainable Chemistry & Engineering

Selecting a green strategy on extraction of birch bark and isolation of pure betulin using monoterpenes. Alexandr E. Grazhdannikov a, Lyubov M. Kornaukhova a, Vladimir I. Rodionov a, Natalia A. Pankrushina a

a

, Elvira E. Shultsa, Anne S. Fabiano-Tixier b,c, Sergey A. Popov*a, Farid Chemat*b,c.

Novosibirsk Institute of Organic Chemistry, Acad. Lavrentyev Ave. 9, Novosibirsk 630090, Russia

b

Université d’Avignon et des Pays de Vaucluse, INRA, UMR408, GREEN Team Extraction, F-84000

Avignon Cedex, France. *

Corresponding authors: [email protected]; Tel: +7 383 3306748; [email protected]; Tel:

+33 0490144465 Keywords: triterpenoids; monoterpenes; extraction; catalytic hydrogenation; green refining, COSMO-RS

Abstract The aim of this work was to study sustainable approaches to pure betulin using green low-polar solvents from natural feedstock – pine monoterpenes and limonene. The conventional methods of extracting outer birch bark (OBB) lead to extracts that contain only 50–75% of betulin, while refining processes of OBB extracts are usually labor-consuming and employ toxic solvents. The extractions of OBB using monoterpenes and their hydrogenated analogs at boiling and microwave-assisted (MWA) conditions turned out to be insufficiently selective and led to a significant uptake of extractants on the depleted bark. To obtain pure betulin, the sequence including energy-saving extraction of OBB with a green solvent ethyl acetate (EtOAc), saponification of the extract, and purification of betulin in limonene or hydrogenated monoterpenes was proposed. The protocol of processing primary extracts with stable monoterpenes –limonene, pinane and hydrogenated turpentine oil ensured separation of pure betulin (95– 97%) with high yields (75–82%) and the effective recycling of extractants (81–87%). The refining of plant

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metabolites in monoterpenes can be regarded as a promising method, successfully competing with the known multi-step processes and methods employing toxic solvents.

Introduction. Extractive compounds from versatile plant material have found numerous applications in the development of novel agents for medicine, ingredients for cosmetics, and food additives.1,2 Pentacyclic triterpenoids of birch bark, especially betulin, have been the focus of scientific and technological interest for several decades.3-5 Certain betulin-derived lupane compounds exerted various valuable pharmacological properties, particularly anti-tumor,6 and anti-inflammatory activity.7 It was shown that in most cases, synthetic derivatives exhibited higher bioactivity levels than the starting betulin.3 Consequently, the recovery of betulin of high purity as a scaffold for synthesis is of great interest for scientific and industrial purposes, although complete elimination of impurities is a challenging technological task. The application of conventional green polar solvents - lower alcohols does not provide selective extraction of betulin. The resulting extracts usually comprise the sum of triterpenoids and significant amounts of polar and low-polar impurities.8 The application of intermediate polarity green solvents leads to an increase in triterpenoid content in extracts and a decrease in energy consumption in the extraction cycle, but also does not result in the recovery of pure betulin.9 Betulin content in extracts generally does not exceed 55–75% depending on the raw material and solvents used. Several methods of isolation and purification of betulin from birch bark extracts have been proposed;10-13 however, most of them are multi-stage, material- and labor-consuming, or employ toxic reagents and solvents. The process of multi-step purification of betulin from polar and non-polar admixtures, using benzene and chloroform, was described recently.10 Another combined refining method comprised an alkaline treatment and separation of impurities from betulin using an aromatic hydrocarbon (xylene).11 With respect to sustainable extraction and refining of plant metabolites, the search of processes using “green” solvents14,15 including ones from biomass feedstock,16 appears to be a steady trend. Over the past ACS Paragon Plus Environment

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decade, a great deal of attention has been paid to replacing of industrial hazardous solvents in extraction of natural raw materials and several bio-based solvents including monoterpenes have been proposed as extractants.17-19 Accessible monoterpenes (limonene, α- and ß- pinenes or monoterpenes of pine turpentine, as well as hydrogenated derivatives offer certain advantages compared to conventional low boiling polar extractants (see Supporting Information, Table S1), and are regarded as promising green solvents.14 Monoterpenes possess high flash points, a low specific heat capacity, and vaporization heat values and they are scarcely water miscible. Accessible monoterpenes were successfully tested as alternative solvents for leaching of lipophilic substrates.16,17,20-24 The use of “green” solvent limonene as a selective extractant of betulin from the birch bark under the action of MW was reported.23 Significant solubility of triterpenoids at a high temperature in limonene, good betulin crystallization on cooling, and the poor extraction of polar impurities allowed the authors to prepare pure betulin from the birch bark. Furthermore, hydrogenated monoterpenes that are stable towards oxidation and action of various reagents may be used alternatively. Thus, hydrogenated turpentine oil (pinane) was employed to extract carotenoids, lipids, and terpenes from different natural raw materials.18 Solvent-free environmentally benign hydrogenation of monoterpenes can be accomplished easily with the aid of heterogeneous catalysis.25 Using monoterpenes for selective “green” isolation and purification of betulin, which is a product of high added value, seems to be of particular interest. The two approaches for obtaining pure betulin were assumed promising. 1. Improving the selectivity of OBB extraction. We expected that leaching bark with monoterpenes can result in preferential betulin extraction in comparison with triterpenic acids, while more polar derivatives (sugars and phenols) would not practically be extracted. 2. Using monoterpenes to produce a pure betulin from concentrates of triterpenoids through separating polar and low polar impurities. A removal of low-polar impurities through crystallizing betulin in ACS Paragon Plus Environment


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monoterpenes and separation of acids and polar substances as their salts can be regarded as a reasonable route to prepare pure betulin from extracts. Thus, the objective of our study was to develop effective and eco-friendly methods to produce pure betulin using “green” monoterpenes through selective extraction of OBB or refining of bark extracts.

EXPERIMENTAL SECTION Material and methods. Plant material. An industrial batch of birch (Betula pendula Roth), comprising the outer bark (110 kg), was obtained from the Tomsk Region, Russia (coordinates: 56°28’18”N; 84°37’50”E). The bark was collected from freshly cut trees (crop age ~60 years). The raw material was preliminary dried at 40–50ºC and comminuted on a knife grinder (IYBG-2, NIOCh, Russia) to obtain pieces ~0.3×2 cm. The averaged sample of comminuted raw material ~600 g was dried in a drying cabinet at 40–50ºC to achieve the residual moisture content of 5.3 ± 1.1%. Reagents and standards. Betulin, betulinic acid (> 98% purity), oleanolic acid (97% purity), lupeol (94% purity), and D-limonene (97% purity) were purchased from Sigma Aldrich Chemie GmbH (Schnelldorf, Germany). Ethyl acetate (EtOAc) of the reagent grade was supplied by Vekton (Russia). Turpentine oil (Pinus Sylvestris) and α-pinene (97%) were purchased from the “RASAL” company (Novosibirsk, Russia) and were fresh distilled prior to conducting the experiments. Water bidistillate from the BE-4 system (NV-LAB, Russia) was used. For the analysis method, LC-MS grade methanol from Vekton (Russia) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (99.5%) from Sigma Aldrich Chemie GmbH (Germany) were utilized. NiRa alloy (Ni 50%, Al 50%) was purchased from Chemical Line (Saint Petersburg, Russia), while Ni skeletal catalyst was prepared according to the work.26


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Hydrogenation of monoterpenes. In a stainless steel autoclave equipped with a propeller stirrer, 160 mL of pinene or 160 mL of turpentine oil and 10 g of freshly prepared Ni skeletal catalyst were placed. The autoclave was purged two times with hydrogen, pressurized to 60 Bar, which was then stirred at 150ºC. After 10h, hydrogen was vented from the autoclave, and the reaction mixture was filtered through a filter paper to obtain saturated monoterpenes mixture. Solubility assessment of triterpenoids in monoterpenes. Computational Method: Theoretical Prediction with COSMO-RS.27,28 In this work calculations of relative solubilities of betulin, betulinic acid and lupeol in monoterpenes and EtOAc were done by implementing the COSMO-RS model on COSMOtherm software (C30 1401, CosmothermX17, COSMOlogic GmbH & Co, KG, Cosmologic, Leverkusen, Germany, see Supporting Information). The predictions were carried at 25°C and at boiling point of each solvent (extraction temperature). The predicted solubilities in D-limonene, α-pinene, and pinane solvents were compared with corresponding solubilities of each triterpenoid in EtOAc. Experimental assessment of solubility of triterpenoids in boiling monoterpenes. A flask containing a batch of betulin (500 mg, 97%+), betulinic acid (100 mg, 97%+) or lupeol (300 mg, 94%) was charged with a batch of fresh distilled solvent (D-limonene, pinane). The initial loads of monoterpenes determined from the preliminary experiments were correspondingly 1000 µL (betulin, betulinic acid) and 100 µL (lupeol). The mixture was stirred under a gentle argon flow and heated to monoterpene boiling point temperature, using an oil bath. The temperature in the mixture was controlled, using a Pt thermocouple. Using a long syringe needle, an appropriate monoterpene solvent was added dropwise directly into the flask through a condenser. After the complete dissolution of solids, the flask was cooled to an ambient temperature. The amount of solvent consumed for dissolution was determined gravimetrically. The solubility of the triterpenoids in monoterpenes (w/w) was calculated:



Stt = mtt/mmt,

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1

where mtt – mass of triterpenoid; mmt – mass of added solvent. Extraction of outer birch bark using monoterpenes (reflux boiling). A flask with a batch of milled bark (5 g) was charged with a fresh distilled solvent (D-limonene, pinane, or hydrogenated turpentine, 50 mL). The mixture was heated at reflux over an oil bath for 3h22 under a gentle argon flow. Once the extraction was complete, the flask was cooled to ~120°C; thereafter which the solvent was quickly filtered, and the bark was pressed on the Büchner funnel. The bark on the filter was immediately weighed to determine the solvent uptake. Water (3×30 mL) was added to the filtrate, and the extractant was hydrodistilled at a reduced pressure. The resulting solid residue was dried at 50°C in a drying oven to obtain the crude extract concentrate. All experiments were performed in triplicates. Microwave-assisted extractions of OBB using monoterpenes. Prior to the MWA extraction, OBB was additionally milled to ~0.5 mm pieces. Extractions were carried out in a monomode microwave reactor, Discover-S Class (CEM Corp., USA), at a frequency of 2.45 GHz, with the regulated power of microwave energy up to 300 W. This procedure was conducted with a continuous control of the sample temperature, overpressure in the microwave vial, dynamics of change of microwave power, and the possibility of stirring the reaction mixture. A vial was charged with OBB (0.5 g) and a monoterpene (limonene, pinene, or turpentine oil, 10 ml). The MWA extractions were carried out for 30 min at the isothermal condition of 150ºC.23 Immediately after the MWA extractions, the bark was removed through filtration (filter paper, calico tissue). The extracts were concentrated to dryness using vacuum hydro-distillation, and dried to remove residual water at 50°C. All experiments were performed in triplicates. Extraction of birch bark with wet EtOAc (3.5% H2O). The preparation of wet EtOAc and extraction of birch bark were performed as described in the previous work9 and is detailed in the Supporting Information. ACS Paragon Plus Environment

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Removal of polar admixtures and triterpenoid acids by alkaline processing of primary birch bark extract. A concentrate prepared using preliminary EtOAc (96.5%) extraction of OBB was used for the experiments. A flask containing a batch of primary extract (24.0 g) and KOH (16.0 g) was charged with the mixture of i-PrOH (240 mL) + H2O (120 mL). The resulting mixture was stirred at reflux for 2h and cooled to room temperature. The mixture was vacuum-filtered through a Büchner funnel (filter paper, calico tissue). The resultant precipitate was dried in a drying cabinet at 50–70°C to obtain a concentrate containing residual alkaline admixtures as a grey powder (17.7 ± 0.6 g). All experiments were performed in triplicates. Monoterpene extraction of extract pre-processed with alkali. A flask with a batch of preliminary alkaline-treated primary concentrate (3.0 g) was charged with a fresh distilled terpenic solvent (Dlimonene, pinane, or hydrogenated turpentine, 30 mL). The mixture was stirred at reflux using a DeanStark distilling trap for 2h. The hot mixture was quickly vacuum-filtered through a Büchner funnel. The filtrate was kept at 5°C for 5h. The sediment was vacuum-filtered and pressed on a Büchner funnel, and the residual solvent was vacuum-hydro-distilled from the sediment. The resulting betulin samples were dried at 50°C to a constant weight. The mother liquor was hydro-distilled at ambient pressure. The monoterpenes were separated from the resulting distillates and checked for admixtures (GC-MS). All experiments were performed in triplicates. Analysis of extracts and monoterpenes. HPLC analysis of extracts were performed as described in the previous work9 (Supporting Information). Analysis of monoterpenes, their hydrogenated products and recycled extractants was performed using a standard GC-MS method (Supporting Information). Statistical analysis. All experiments on extraction and purification were performed in triplicates. Extraction yields and content of components were expressed as mean ± SEM. The significance of



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difference between the means was analyzed using the Student test. Test p values < 0.05 were considered statistically significant. Statistical analysis was performed using Statistica, software version 6.0. Results and discussion Hydrogenation of pinene and turpentine oil. Hydrogenation of double bonds of unsaturated monoterpenes results in the formation of saturated green solvents that are stable when it comes to oxidation and action of versatile reagents and drastic processing conditions.24 The hydrogenated derivatives are less polar comparing their precursors; thus, they may be more selective in solublization and separation of solutes of different polarity. Preparation of hydrogenated products using heterogeneous catalysis in solvent-free conditions is in a good agreement with green chemistry principles. Hydrogenation of a neat pinene mixture over NiRa catalyst at 150ºC resulted in a mixture of cis- (predominantly) and trans-pinane (ca 21:1, GC-MS data, Fig. S4, Fig. S5, Supporting Information). The main components of the turpentine used in our hydrogenation experiments were α-pinene (predominantly) and β-pinene mixture (74.2%), 3-carene (22 %), limonene, terpinene and terpinolene traces. According to GC-MS data (Fig. S5, Supporting Information) the major components of the hydrogenated turpentine oil were as follows: trans pinane (3.1%), cis pinane (72.0%), and 1,1,4- trimethylcycloheptane (TMCH) (6.7%), transmenthane (1.4%), 1,4 cis-menthane (1.4%), carane (13.7%) resulting from carene.29,30 The products of hydrogenation were isolated in almost quantitative yields.

COSMO-RS predicted and experimental solubility assessment of triterpenoids in monoterpenes. The results of COSMO-RS calculations and experimental data were used as decision tools for selecting monoterpenes as extractants of OBB triterpenoids. Table 1 shows the COSMO-RS results predicted solubilities of major OBB triterpenoids in monoterpenes and EtOAc (reference solvent) expressed in log10(x solub) (best solubility is set to 0). The predictions


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were carried out at boiling point of each solvent, which was the temperature of extraction under laboratory conditions and at 25°C (details are given in Supporting Information).

Table 1. COSMO-RS results of solubility prediction of triterpenoids in EtOAc and monoterpenes

Predicted solubility at 25°C, log10(x solub) Triterpenoid

Pinane

Predicted solubility at b.p., log10(x solub) Ethyl

Limonene

Pinane

Ethyl

D-

α-

acetate

Limonene

Pinene

25°C

25°C

25°C

25°C

77°C

176°C

155°C

169°C

Betulin

-3,5261

-5,1509

-5,4019

-5,5651

-2,3934

-1,5673

-1,9643

-1,8362

Betulinic acid

-2,7143

-3,6509

-3,9519

-4,1444

-1,797

-1,0166

-1,3726

-1,2827

Lupeol

-2,9276

-3,3582

-3,4163

-3,4775

-1,9917

-0,9616

-1,1714

-1,0529

acetate

αPinene

x solub – solute/solvent ratio expressed as a mole fraction Grey colour: Reference; Green colour: Better or equivalent to reference; Red colour: Worse than reference. The predicted solubilization indexes of boiling monoterpenes for triterpenoids were substantially higher than that of the reference solvent, whereas at room temperature they were found several orders of magnitude lower. We also assessed the solubization of pure betulin, betulinic acid and lupeol in limonene and pinane at the boiling point of monoterpenes. According to experimental data, the solubilities of triterpenoids in boiling monoterpenes were very high: 19 ± 3% w/w (limonene) 16 ± 3% (pinane) for betulin; 5.1 ± 0.6% w/w (limonene), 3.3 ± 0.4% (pinane) for betulinic acid; 155±25% w/w (limonene), 135±25% w/w (pinane) for lupeol (Table S3, Supporting Information). The perfect solubility of lupeol in monoterpenes is in agreement with COSMO-RS results and indirectly consistent with the known data on good extraction of



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lupeol by hydrocarbons.31 Thus, in accordance with calculation and experimental data, monoterpenes appear to be promising solvents for extracting triterpenoids.

Selection of monoterpenes for extraction and processing of triterpenoids. When the bark was extracted using limonene, pinene or turpentine oil, similar inherent triterpenoid patterns and yields of extracts were obtained. Along with that, a substantial drawback of using pinene and turpentine oil was the accumulation of artifact impurities, both in concentrated extracts and in recycled solvents. At the same time, the formation of impurities in extraction and subsequent processing of limonene (relatively stable monoterpene) extracts was insignificant. To avoid solvent degradation, we studied the use of more stable extractants - hydrogenated monoterpenes obtained from turpentine and pinene. Extraction of outer OBB using limonene and hydrogenated monoterpenes. Processing of OBB with high and medium polarity solvents (EtOH, EtOAc) usually results in extracts comprising mixtures of triterpenoids, and polar and non-polar impurities. We assumed that the extraction of OBB with monoterpenes would result in extracts containing lower amounts of triterpenic acids and polar impurities.23 In the present study, the preliminary examination revealed a relatively high content of triterpenic acids and a low content of lupeol in the extracts (EtOAc) of the outer bark. Thus, according to HPLC data, the extract of the outer OBB with wet EtOAc contained triterpenic acids ~ 9% and lupeol only ~ 2.5%. The removal of easily crystallizable triterpenic acids and polar impurities affecting the color of the product are the most complicated issues in a process of betulin purification. The extraction of OBB with limonene, pinane, and hydrogenated turpentine under boiling conditions resulted in extracts that were very similar in composition (Table 2).


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Table 2. Primary extraction of external birch bark using monoterpenes and EtOAc (3.5% H2O). Extract

Extractant

yield, %

Solvent

Content of the component (component group) in

uptake,

the extract, according to HPLC, % w/wc

w/w

Polar

Triterpeniс

bark

admixtures

acids

Betulin

Lupeol

D-limonenea

31.8±2.9

1.9±0.2

6.7±0.4

8.4±0.6

65.5±3.3 2.9±0.2

D-limoneneb

32.0±3.5

1.8±0.3

6.4±0.5

8.6±0.3

66.3±3.0 2.9±0.3

pinanea

25.7±3.0

2.0±0.2

4.9±0.2

7.0±0.3

62.4±2.4 2.8±0.2

pinaneb

32.5±2.1

2.0±0.2

4.5±0.3

6.1±0.4

68.1±2.2 3.1±0.2

turpentine oil

26.3±2.1

2.1±0.2

5.2±0.5

7.3±0.5

65.1±3.1 3.0±0.4

30.0±0.5

2.7±0.2

15.2±1.7

9.0±0.4

61.6±2.4 2.6±0.3

hydrogenateda (EtOAc wet 96.5%)a

a

Reflux boiling extraction

b

Microwave assisted extraction

c

Calculated as area ratio of the component peak (component peaks) to the total integrated area

All data are given as average ± standard error. Unlike EtOAc extractions, a relatively low content of polar impurities was detected in the extracts for all extraction experiments using monoterpenes, (Fig. 1).



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Fig 1. HPLC data obtained with DAD at 210 nm for extracts of birch bark employing different extractants. Below: HPLC chromatogram of outer birch bark with pinane (reflux boiling). Top right: Comparison of magnified “polar” sections of chromatograms: extracts with wet EtOAc (96.5%); D-limonene, “pinane” mixture (reflux boiling). P- “polar” compounds; TTA- triterpenic acids (betulinic+oleanolic); B-betulin; L-lupeol Unlike pinene and turpentine oil, the saturated monoterpene solvents were stable in extraction and recycling conditions. No admixtures were accumulated in saturated solvents, and this allowed their comprehensive recycle. In the reflux boiling experiments, the application of limonene led to somewhat higher yields of extracts, which is in agreement with COSMO-RS prediction results. Since a good selectivity on betulin extraction with limonene was reported,23 we performed MWA testing experiments to study the selectivity of OBB extraction using limonene and pinane. Our extraction experiments in MWA conditions (150°C, 30 min) resulted in extracts, which were similar in compositions to those obtained using boiling monoterpenes. In general, the use of MWA extraction in monoterpenes allowed reducing time of extraction and led to a certain yield increase compared to the conventional OBB boiling in limonene and pinane (Table 2). Compared to the corresponding values obtained for extraction in EtOAc, a lower but still significant content of triterpenic acids and polar admixtures was found for monoterpene extracts. Thus, using monoterpenes did not ensure sufficient selectivity improvement of ACS Paragon Plus Environment

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betulin extraction from the bark. To evaluate the recycling efficiency, we estimated the costs of extractant recovery both from miscella and from the depleted bark cake. Specific energy consumptions for evaporation of monoterpenes were calculated using specific heat and latent vaporization heat values. The heat expenses on evaporation of monoterpenes were found to be only ~10% higher than the same for EtOAc. At the same time, the noticeable miscella uptake on the extracted bark and high boiling points of monoterpenes (169ºC, pinane, 176ºC, limonene) should be considered. The residual monoterpene extractant cannot be removed from the depleted material by direct heating, while hydrodistillation of monoterpenes is more energy consuming compared to the same recycling of low-boiling solvents of intermediate polarity. Unlike EtOAc azeotrope, comprising only ~8% w/w water, the azeotropes of limonene and pinane contain ~62% w/w water. Thus, the costs for solvent removal are 713 kJ / kg, 4296 kJ / kg, 4274 kJ / kg, respectively, for EtOAc, limonene, and pinane as aqueous azeotropes. Consequently, except for a low content of polar admixtures and a certain increase in betulin percentage, the extraction of OBB with limonene, hydrogenated monoterpenes provides no advantages over the processes employing more accessible green solvents - ethanol and ethyl acetate. A relatively high price of extractants and notable regeneration expenses, as well as insufficient selectivity on the target component, betulin, are the limitations of monoterpenes usage for bark extraction. In this regard, we studied other possibilities for sustainable refining using monoterpenes, concretely, the purification of primary extracts and pure betulin production.

Re-extraction of the primary triterpenoid extract using limonene and hydrogenated monoterpenes. An interesting property of triterpenoids is their high solubility in monoterpenes at an elevated temperature and its sharp drop at a decreased temperature (COSMO-RS prediction). We assumed that the secondary extraction of concentrates employing monoterpenes followed by crystallization could be used in the betulin purification technology. The concentrate comprising the mixture of triterpenoids obtained in EtOAc (3.5% H2O) extraction was used as a primary extract since the EtOAc green extraction of OBB ACS Paragon Plus Environment


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provides the effective recovery of triterpenoids and a complete low-cost recycling of the solvent.9 With respect to a high added value of purified betulin, we have studied the possibility of refining of primary concentrates with green solvents - monoterpenes to separate the undesirable impurities.

Table 3. Processing of primary extracts (wet EtOAc extraction of the outer birch bark) by re-extraction with terpene hydrocarbons.

Re-extractant

Yield, %

Content of the component (component group) in

counting

the extract, according to HPLC, % w/wa

on initial

Polar

Triterpeniс

extract

admixtures

acidsb

limonene

95.5±3.2

4.2±0.4

7.8±0.4

66.4±3.2 3.2±0.3

pinane

92.4±2.1

3.8±0.4

6.9±0.6

63.3±4.2 3.1±0.4

Turpentine oil

92.8±3.7

4.1±0.3

7.1±0.4

65.6±3.8 3.3±0.3

Betulin

Lupeol

hydrogenated

a

Calculated as area ratio of the component peak (component peaks) to the total integrated area

b

Predominantly betulinic acid+oleanolic acid

All data are given as average ± standard error

When the concentrates of primary extracts (wet EtOAc) were heated in monoterpene hydrocarbons, nearly complete dissolution of solids took place. The data in Table 3 shows that the separation of insoluble admixtures by hot filtration led only to a slight decrease of polar impurities and triterpene acids content in the extracts.


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Processing the primary extract using saponification and treatment in monoterpenes. To improve separation of triterpenic acids and phenols, we studied the conversion of polar impurities into their salts (which are completely insoluble in hydrocarbons). We found that treating the extracts with water-alkaline solutions and simultaneously removing the water as azeotropes with low-polar solvents (xylene, monoterpenes) did not result in a notable separation of triterpenic acids salts from the extract. Salts of weak triterpenic acids are poorly soluble in water and are hydrolyzed readily in aqueous solutions upon formation. In an aqueous phase, the equilibrium between weak acids and their salts shifts towards formation of acids that are readily soluble in a hot hydrocarbon phase used in this process. Therefore, when employing aqueous-alkaline treatment and simultaneous azeotrope distillation, the conversion of TTA to salts at their high content in the extract was found to be incomplete. Consequently, an alternative method of removal of triterpenic acids is necessary. Inorganic salts of betulinic acid are highly soluble in aqueous-alcoholic solutions (~ 30% H2O) of strong inorganic base, whereas betulin is poorly soluble in such solutions. We used a saponification with an alkaline water-alcoholic solution for the removal of a major amount of polar impurities and triterpene acids. The resulting semi-product (precipitate) contained about 3% of the TTA (as salts), ca 1.5–2% lupeol, ca 3% polar impurities, 83-85% betulin. Upon separation of the alkaline filtrate, the resulting precipitate was treated with hot monoterpenes (limonene, pinane, or hydrogenated turpentine oil). The typical HPLC data of the initial and purified extracts are shown in Fig. 2.



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Fig 2. HPLC data obtained with DAD at 210 mm for birch bark (OBB) extract employing different extractants and processing. Top (1): HPLC chromatogram of extract prepared by re-extraction of OBB extract with boiling hydrogenated turpentine oil (HTO). Middle (2): HPLC chromatogram of betulin sample prepared from OBB extract (EtOAc-H2O) by preliminary washing in boiling i-PrOH-H2O-KOH solution and cold filtration. Below(3): HPLC chromatogram of betulin sample prepared from OBB extract: 1. preliminary washing in boiling i-PrOH-H2O-KOH solution and cold filtration. 2. boiling in HTO, hot filtration, crystallization. P- “polar” compounds; TTA- triterpenic acids (betulinic+oleanolic); B-betulin; L-lupeol.

Boiling in the monoterpenes resulted in the dissolution of betulin and low-polar impurities, while highpolar admixtures and acid salts were insoluble. Betulin crystallized perfectly from the monoterpene solutions upon cooling to room temperature; at the same time, mother liquors were enriched with lowpolar admixtures, preferably lupeol which is better soluble in hydrocarbons25 (Table 4).


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Table 4. Isolation of betulin from EtOAcaq primary extracts treated with base using re-extraction with monoterpene hydrocarbons (limonene or hydrogenated monoterpene). Content of the component (component group) in the extract, according to HPLC, % w/wb

Crystalline Extractant

phase

crystals

betulin

Polar

Triterpeniс

admixtures

acids

Betulin

Mother liquor Lupeol

Betulin

Lupeol

a

yield, % limonene

75,5±2.0

0.2±0.01

0.09±0.015 96.8±0.5 0.20±0.021

49.5±0.5

20.2±0.7

82.1±3.5

0.2±0.01

0.11±0.012 94.9±0.5 0.22±0.015

37.1±0.4

25.2±0.5

81.5±2.6

0.2±0.01

0.10±0.012 97.0±0.3 0.12±0.015

44.2±0.7

23.7±0.6

pinane

HTO

a

counting on betulin content in initial EtOAc-H2O extract

b

Calculated as area ratio of the component peak (component peaks) to the total integrated area.

The resulting betulin had a perfect white color due to the absence of staining impurities. Despite the relatively high content of betulin in the mother liquors, the losses of betulin were insignificant, since only minor portions of betulin were contained in the mother liquors and the yield of the crystalline phase was 8 to 10 times higher than the yield of the concentrate from the mother liquor. Hydrodistillation of mother liquors allowed recuperation of 81–87% of the monoterpene solvent, which can be recycled. Residual amounts of the monoterpene solvent (0.5–2% w /w) were removed from crystalline betulin by vacuum hydrodistillation. An operation with an additional washing or crystallization from ethyl acetate is also feasible for the complete removal of monoterpenes. The high yield and purity of betulin were ensured by the effective and selective extraction of betulin from pre-processed mixtures of triterpenoids containing other natural impurities harnessing a hot monoterpene ACS Paragon Plus Environment


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solvent in which betulin was readily soluble in contrast to polar impurities. As the temperature lowered, betulin crystallized perfectly, while low-polar impurities were separated with monoterpene mother liquor.

Fig 3. Comparative assessment of heat expenses on OBB extraction and purification of betulin

The results of specific heat consumptions for extraction, purification and solvent recycle calculated using specific heat and latent vaporization heat values for corresponding solvents (solvent mixtures) at the each step are given on the Fig 3. Regarding the purification in monoterpenes and their recycle, the highest corresponding values (limonene) were used for the estimation. The comparative expenses on conventional crystallizing the same amount of the extract from 95% EtOHaq and solvent recycle were calculated (the high costs of EtOH extraction and recycle from the processed OBB,9 in this case were not taken into account). It should be mentioned, that a single crystallization from EtOH cannot provide product of 95%+ purity.13 The high solubilities of betulin in hot monoterpenes allow low specific solvent consumption for purification of betulin. In addition, monoterpene hydrocarbons possess low evaporation heat values, therefore the proposed scheme has lower heat expenses (54710 vs 101738 kJ/kg) as compared to the conventional crystallization using EtOHaq (the low solubility of betulin in EtOH, high costs of regeneration of polar solvent). The major expenses are ones attributed to the regeneration of aqueous iPrOH after the alkaline treatment. This step can be further optimized, in particular by repeated using of the


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same alkaline solution (a high capacity with respect to acidic impurities) or isopropanol recovery as aqueous azeotrope. The material flowsheet of OBB extraction with a low boiling solvent (EtOAc aq.) and isolation of pure betulin from the extract using monoterpene-derived solvents is given in the Supporting Information (Fig. S7). The best yield and purity of betulin was obtained using HTO, while application of limonene resulted in a lower yield, and the use of pinane provided higher lupeol content. The application of readily available HTO for the purification of betulin appears to be advantageous, since a mixture of stable saturated hydrocarbons dissolves betulin poorly upon cooling, but solubilizes low-polar impurities somewhat better compared to pinane. Thus, the treatment of the saponified extract in monoterpene solvents is a highperfomance alternative for chromatographic purification of betulin from non-polar and residual polar impurities. The composition of turpentine oils used for hydrogenated monoterpenes production can vary depending on pines species and their origination. Thus, P. pinaster turpentine oils contain mainly pinenes mixtures (up to 97%).32 Turpentine obtained from P. sylvestris (Russia), contains generally 60–77% of the mixture of pinenes and 3-carene (up to 27%).33 For P. sylvestris turpentine originated from Turkey pines, 50–70% of pinenes and 6–10% of 3-carene content were reported.34 The compositions of resulting hydrogenated solvents may be different depending on the starting turpentine oil, hydrogenation conditions and catalyst type, albeit the major components would always be pinanes. Apparently, regardless of the type of pine turpentine used, the composition of hydrogenated monoterpenes would not dramatically affect the technological parameters of the process for betulin purification. It’s notable that the preparation of monoterpenes mixtures devoid of sesquiterpenes and oxygenated monoterpenes can be accomplished by distillation; therefore, the use of turpentine oils of a different origin and composition for the production of green solvents and their effective use seems promising. Conclusions. In this work, the approaches to prepare pure betulin by the direct extraction of birch bark (OBB) or by processing OBB extracts using green low polar solvents: pinene, pine turpentine oil, products ACS Paragon Plus Environment


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of their hydrogenation, and limonene, were studied. According to the experimental and computational data (COSMO RS) on solubilization in limonene and pinane, monoterpenes were assumed promising extractants of major triterpenoids of OBB. It was found that using monoterpenes to treat OBB under reflux boiling and MWA conditions, extracted a sum of triterpenoids with the insignificant leaching of polar admixtures. It was shown that the insufficient selectivity of extraction and high costs of solvent recycling from the processed OBB impose restrictions on the direct usage of monoterpenes in the preparation of betulin. The sequence including energy saving extraction of OBB with aqueous EtOAc, saponification of the extract, and purification of betulin in limonene or hydrogenated monoterpenes was proposed. An efficient green protocol for preparing of pure betulin (95–97%) from extract with high overall yields (75–82%) and with efficient solvent recycling 81–87% was elaborated. The efficiency of separation of high-polar impurities is ensured through their preliminary conversion to salts that are completely insoluble in monoterpenes, while low-polar impurities are withdrawn by crystallizing betulin in monoterpenes. The application of hydrogenated monoterpenes, pinane and hydrogenated turpentine in the processing of extracts excluded accumulation of artifact impurities and enabled the effective recycling of solvents. Employing green monoterpene solvents in the process of refining triterpenoids is assumed as a prospective and energy saving alternative to methods using toxic solvents and labor-consuming chromatographic steps. A further study of green low-polar solvents from natural feedstock for the extraction and purification of individual plant metabolites seems promising, since such technologies may be competitive in terms of economic viability and sustainability. ASSOCIATED CONTENT Supporting Information. Properties of monoterpenes. Method of OBB extraction using wet EtOAc(3.5%) Methods of HPLC analysis of extracts, method of GC-MS analysis of monoterpenes. Material on COSMO-RS solubility calculation results. ACS Paragon Plus Environment

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Experimental data on azeotrope composition of monoterpenes. GLC data on composition of monoterpenes. Experimental data on solubility of betulin and betulinic acid in monoterpenes. The material flowsheet of green OBB extraction and betulin refining. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION ∗Corresponding authors: [email protected]; Tel: +7 383 3306748; [email protected]; Tel: +33 0490144465 The manuscript was written through contributions of all authors. The authors declare no conflict of interest and all have given approval to the final version of the manuscript.

Acknowledgements. This research was financially supported by the Joint Basic Research Project #12 of Belarus National Academy of Sciences and Russian Academy of Sciences and by the Russian Foundation of Basic Research (Grant 18-53-76001, Application Nr. RUS_ST2017-139). Analytical and spectroscopic studies were performed at the Chemical Service collective Center of the SB RAS.

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Keywords: birch triterpenoids; monoterpenes; catalytic hydrogenation; betulin green refining TOC Synopsis: Recyclable hydrogenated pine monoterpenes and limonene effectively replaced petroleumbased solvents in the isolation of pure betulin from bark extracts.


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HPLC data obtained with DAD at 210 nm for extracts of birch bark employing different extractants. 60x43mm (600 x 600 DPI)



HPLC data obtained with DAD at 210 mm for birch bark (OBB) extract employing different extractants and processing. 60x43mm (600 x 600 DPI)


Page 26 of 27

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Comparative assessment of heat expenses on OBB extraction and purification of betulin 60x42mm (600 x 600 DPI)


Selecting a green strategy on extraction of birch bark and isolation of

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