Betulin Content Research Articles

Volatile matter characterization of birch biochar produced under pressurized conditions

AbstractThe volatile matter (VM) content and composition of birch biochars produced at 320 °C under elevated pressure (0.1–11 MPa) and constant pressure or constant volume reactor conditions were characterized by thermogravimetry/mass spectrometry (TG/MS) and pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS). Some of the thermal properties of the biochars and the composition of the VMs varied as a function of the maximal pressure applied during carbonization. The samples prepared at higher pressures released more volatiles up to 320 °C, while the maximal rate of thermal decomposition at around 440 °C showed decreasing tendency with the carbonization pressure. In terms of VM composition, the most apparent effect was the significant increase of the amounts of apoallobetulins from biochars prepared at elevated pressures, which were formed by dehydration, ring closure and rearrangement from the betulin content of birch. The change in the ratio of the evolved guaiacol and 4-methylguaiacol as well as that of syringol and 4-methylsyringol as a function of the maximal pressure of carbonization indicated a modification of the lignin decomposition mechanism.

STUDY OF EXTRACT SUBSTANCES OF WHITE BIRCH BARK

The article presents the results of a comparative study of extractive substances and the main substance - betulin content of Turkestan white birch bark, as well as the results of the study of its structure and some physico-chemical parameters. Betula Turkestanica Roth extractives contain 82% betulin.

Инженерная методика расчета установки извлечения бетулина из бересты березы

The analysis of the current state of the process of betulin extraction from birch bark has shown the relevance of the implementation of periodic extraction technology using toluene as a solvent at small enterprises of the timber industry complex. This article presents the scheme of an extraction plant consisting of an extractor, an evaporator, a condenser, a florentine flask, an extract collector, as well as the principle of the plant operation. The extraction process is carried out in two stages: when the raw material is at rest in relation to toluene and when the raw material is extracted by continuous feeding of fresh extractant. The engineering procedure for calculating the extractor has been developed, allowing to determine its overall dimensions (diameter and height) and the duration of individual stages of the extraction process. The duration of the first stage has been calculated by solving Fick’s differential equation of mass conductivity under boundary conditions of the first kind. The duration of the second stage has been calculated considering the fact that betulin extract is continuously withdrawn from the extractor and fresh extractant is injected. In the latter case, the betulin content in the extractant varies not only in time, but also with the height of the extractor. The developed mathematical description and subsequent modeling made it possible to obtain the calculated curves of betulin distribution in birch bark at different points in time, as well as to compare the experimental and calculated data on the kinetics of the average betulin content in birch bark and toluene for each stage. Meanwhile, the kinetic curves of the average betulin content in birch bark at the second stage are presented for different heights of the extractor. It has been established that the optimal duration of the first stage is 20 min, and with the continuous feeding of fresh extractant with a flow rate of 22.5 kg/h at the second stage, the process speed increases by 6 times. This significantly reduces the total extraction time under the specified process parameters. The necessity for the introduction of the third stage (the stabilization period of the betulin content over the layer height and the cross– section of particles) has been shown.

Multi-component content determination of Dracocephalum tanguticum by quantitative analysis of multi-components by single-marker

This study aims to establish a method for the simultaneous determination of 7 active components in Dracocephalum tanguticum and to evaluate the quality of medicinal materials from different habitats. The method was established with high performance liquid chromatography(HPLC) and the gradient elution was performed with the mobile phase of acetonitrile-methanol-0.2% phosphoric acid solution at a column temperature of 35 ℃, an injection volume of 15 μL, and a flow rate of 0.6 mL·min~(-1). The detection wavelength was set as 215 nm. With rosmarinic acid as the internal reference, the relative correction factors and the content of other 6 components were calculated. The results were compared with those obtained with the external standard method. The results showed that the samples from Huangzhong county, Qinghai province had the best quality, with the highest content of p-hydroxybenzoic acid, cosmosiin, rosmarinic acid, oleanolic acid, and ursolic acid(9.29, 12.14, 6.02, 3.11, 17.67 mg·g~(-1) respectively). The samples from Chaya county, Tibet autonomous region ranked the second, with the highest content of betulin and betulinic acid(15.53, 7.17 mg·g~(-1), respectively). The method is accurate, reliable, and repeatable and suitable for the simultaneous determination of multiple components in D. tanguticum. The content of functional components varied in the samples from different producing areas and can be used as the indicator for the quality evaluation of medicinal materials.

Extractable Compounds in a Birch Tree—Variations in Composition and Yield Potentials

Extractives from silver birch (Betula pendula) can play an important role in the future bioeconomy by delivering the feedstock, for instance, for antioxidative applications. It is, therefore, inevitable to gain knowledge of the distribution of extractive content and composition in the different tissues of the tree for estimating the potential volumes of valuable extractable compounds. This study examines the extractable compound distribution of different tree tissues such as outer and inner bark and wood, respectively, considering the original height of the stem and comparing the yields after Soxhlet and accelerated solvent extraction (ASE). Eleven parts of the model tree (seven stem discs and four branches) were separated into primary tissues and extracted with a ternary solvent system. The investigated extraction methods resulted in a comparable performance regarding yields and the composition of the extractives. The extractives were divided into single compounds such as betulin, lupeol, γ-sitosterol, and lupeone and substance groups such as carbohydrates, terpenes, aromatics, and other groups. The distribution of single substances and substance groups depends on the location and function of the examined tissues. Furthermore, the evidence for the correlation of a single substance’s location and original tree height is stronger for lupeol than for betulin. Primary betulin sources of the calculated betulin output are the outer bark of the stem and the branches. By using small branches, further potential for the extraction of betulin can be utilized. A model calculation of the betulin content in the current birch tree revealed a significant potential of 23 kg of betulin available as a valuable chemical resource after by-product utilization.

Betulin‐containing bioactive amphiphilic copolymers: Synthesis, characterization and self‐assembly

AbstractBetulin, a natural triterpene possessing a wide range of biological activities, has been modified via Steglich esterification to produce a methacrylate derivative capable of participating in radical polymerization. The modification is intended to increase the solubility of betulin, which is almost insoluble in water, by incorporating into the composition of an amphiphilic copolymer. The resulting solubility of betulin in water increased from 8·10−5 mg/mL to more than 10 mg/mL per betulin. For this, well‐defined biocompatible thermoresponsive copolymers with high betulin content have been synthesized through the conventional and RAFT copolymerizations of betulin methacrylate (BeMA) with methoxy oligo(ethylene glycol) methacrylate (MOEGMA). BeMA was more reactive in the BeMA‐MOEGMA pair; the resulting copolymers were slightly enriched with BeMA units (r1 = 1.70, r2 = 0.93). The copolymers exhibited LCST‐type behavior. LCST was tuned by balancing the hydrophobic betulin and hydrophilic OEG fragments. The molecular weight characteristics of polymers and their behavior in aqueous and organic solutions have been studied. Copolymers obtained by conventional polymerization had lower cloud points than RAFT copolymers of the same composition. The copolymers are characterized by low critical micelle concentrations that decrease as the BeMA fraction in the copolymers increases. RAFT copolymers were shown to form unimolecular micelles resistant to dissociation, which is promising for their use in targeted drug delivery.

The Study of Betulin Particles Containing Hydrogels Prepared by Antisolvent Precipitation

The aim of the study was to investigate the effect of process parameters on the production of hydrogels with antisolvent precipitation using unpurified (UB) and purified (PB) birch outer bark betulin powder samples. Experimental activities are focused on the production of hydrogels using concentration of colloidal dispersions with filtration of UB or PB sample particles obtained by antisolvent precipitation method varying the dilution of saturated (at boiling temperature) ethanol solutions with different water content. During the study the maximum solubility in ethanol at boiling point of PB (22.0 g/L) and UB (55.0 g/L) was determined. For obtaining gel with the highest liquid content (absorption capacity 37.45 g per 1 g of dry matter) from PB by antisolvent precipitation the optimal saturated ethanol solution dilution with water was 12.5 vol%. In the case of UB hydrogel, by dilution of ethanol from 25 to 10 vol%, a gradual decrease of hydrogel dry matter yield and a simultaneous increase of the filtrate dry matter yield can be observed, which is related with leaching of the particles through the filter paper together with a solution and form a colloidal suspension of particles. For obtaining gels with high liquid content from UB the optimal saturated ethanol solution dilution with water was in the range from 20.0 to 12.5 vol% (absorption capacity in average 18 g per 1 g of dry matter). Comparing the chemical compositions of the raw materials and dry matter of hydrogels in the case of PB composition remains quite similar while in UB dry matter composition the betulin content increased from 52 to even 69 wt%. PB at the optimal dilution of 12.5 vol% of ethanol managed to achieve average particle size - 231.7 nm and with UB in the optimal dilution range from 20 to 12.5 vol% the range of average particle size was from 304.7 to 189.8 nm.

Epigenetic modification associated with climate regulates betulin biosynthesis in birch

The Betula genus contains pentacyclic triterpenoid betulin known for its environmental adaptation and medicinal properties. However, the mechanisms underlying betulin biosynthesis responding to climate change remain unclear. In this study, the role of epigenetic modification (DNA methylation) in betulin biosynthesis was examined and how climatic factors influence it. Whole-genome bisulfite sequencing was performed for greenhouse-grown Chinese white birch (Betula platyphylla Sukaczev) treated with DNA methylation inhibitor zebularine (ZEB) and a natural birch population in Northeast China. ZEB treatment significantly affected the CHH methylation level of transposable elements and betulin content in a hormesis dose-dependent manner. The methylation and expression of bHLH9, a key transcriptional factor controlling betulin biosynthesis, were also consistently affected by ZEB treatment as a hormetic dose–response. In the natural population, there was a positive correlation between promoter methylation of bHLH9 and summer precipitation, while winter temperature was negatively correlated. Thus climate-dependent methylation of bHLH9 regulates the expression of downstream genes involved in betulin biosynthesis. This study highlights the role of environmental signals to induce epigenetic changes that result in betulin production, possibly helping to develop resilient plants to combat ongoing climate change and enhance secondary metabolite production.

Evaluation of Antioxidative Mechanisms In Vitro and Triterpenes Composition of Extracts from Silver Birch (Betula pendula Roth) and Black Birch (Betula obscura Kotula) Barks by FT-IR and HPLC-PDA.

Silver birch, Betula pendula Roth, is one of the most common trees in Europe. Due to its content of many biologically active substances, it has long been used in medicine and cosmetics, unlike the rare black birch, Betula obscura Kotula. The aim of the study was therefore to compare the antioxidant properties of extracts from the inner and outer bark layers of both birch trees towards the L929 line treated with acetaldehyde. Based on the lactate dehydrogenase test and the MTT test, 10 and 25% concentrations of extracts were selected for the antioxidant evaluation. All extracts at tested concentrations reduced the production of hydrogen peroxide, superoxide anion radical, and 25% extract decreased malonic aldehyde formation in acetaldehyde-treated cells. The chemical composition of bark extracts was accessed by IR and HPLC-PDA methods and surprisingly, revealed a high content of betulin and lupeol in the inner bark extract of B. obscura. Furthermore, IR analysis revealed differences in the chemical composition of the outer bark between black and silver birch extracts, indicating that black birch may be a valuable source of numerous biologically active substances. Further experiments are required to evaluate their potential against neuroinflammation, cancer, viral infections, as well as their usefulness in cosmetology.

차가버섯 추출물의 저장조건에 따른 기능성분 변화 및 품질안정성

This study investigated the effect of temperature on functional constituents and quality stability in chaga mushroom extract during an eight-week storage period. Soluble solid and total polyphenol content (26 mg/mL) showed no significant changes during storage, whereas pH values increased from 5.38 to 5.46 (p<0.05). DPPH radical-scavenging activity exceeded 70% regardless of storage conditions; in contrast, ABTS radical-scavenging activity declined by 6.2%, 7.1%, and 7.9% after eight weeks of storage at 25°C, 35°C, and 45°C, respectively. The β-glucan content (2.58%) was stable at −20°C and 25°C, but it significantly declined after 6 weeks of storage at high temperatures (p<0.05). Similarly, the betulin content (12 μg/mL) was constant at −20°C. Microbial growth was not detected in the extract at any point during the storage period, owing to heat treatment (95°C for 10 min). Overall, the results on functional constituents and quality stability of chaga mushroom extract suggest that storage conditions may affect the chemical attributes of the extract.

Putrescine Promotes Betulin Accumulation in Suspension Cell Cultures of Betula platyphylla by Regulating NO and NH4+ Production

Putrescine (Put) can enhance secondary metabolite production, but its intrinsic regulatory mechanism remains unclear. In this study, Put treatment promoted betulin production and gene expression of lupeol synthase (LUS), one of betulin synthetic enzymes. The maximum betulin content and gene expression level of LUS was 4.25 mg·g−1 DW and 8.25 at 12 h after 1 mmol·L−1 Put treatment, approximately two- and four-times that in the control, respectively. Put treatment increased the content of nitric oxide (NO) and its biosynthetic enzyme activity of nitrate reductase (NR) and NO synthase (NOS). Pretreatment of the birch suspension cells with NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- 1-oxyl-3-oxide (cPTIO), NR inhibitor sodium azide (NaN3), and NOS inhibitor NG-nitro-L-Arg methyl ester (L-NAME) decreased Put-triggered NO generation and blocked Put-induced betulin production. Put treatment improved the content of NH4+ and its assimilation enzyme activity of glutamate synthase and glutamate dehydrogenase. NH4+ supplementation also promoted NO and betulin production. Thus, the above data indicated that Put-induced NO was essential for betulin production. NO derived from NR, NOS, and NH4+ mediated betulin production in birch suspension cell cultures under Put treatment.

Cloning, expression characteristics of a new FPS gene from birch (Betula platyphylla suk.) and functional identification in triterpenoid synthesis

Pentacyclic triterpenoids from birch (Betula platyphylla suk.) display a broad range of pharmacological activities and have potential as anti-cancer and anti-AIDS drugs. FPS (Farnesyl pyrophosphate synthase) gene from birch has been cloned and its function in triterpene synthesis has been identified firstly in this paper. It was named BpFPS. In this study, the expression of BpFPS in birch leaves and bark induced by MeJA (Methyl Jasmonate) and SA (Salicylic Acid) was detected by fluorescence quantitative PCR, and the cloning and cis acting elements of BpFPS promoter were analysed. It was clear that it has binding site of MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factors, temperature responsive element, light cis acting regulatory element, and auxiliary responsive element. The overexpression of BpFPS in yeast promoted the increase of squalene content of triterpenoid precursor, and the expression of BpFPS in birch promoted the significant increase of betulinic acid, oleanolic acid and betulin content, which were 1.081, 4.064 and 2.191 times higher than that of the wild birch respectively. The results showed that the FPS gene of birch was cloned for the first time. It is the key gene in the MVA synthesis pathway and plays an important role in triterpenoids synthesis. The acquisition of this new gene and the identification of its function will contribute to the efficient synthesis of triterpenoids in birch or yeast by genetic engineering and synthetic biology in future.

Changes in Composition of Birch Outer Bark Extractives After Recrystallization with C2-C5 Alkanols

The effects on birch outer bark (BOB) ethanolic extractives’ chemical composition after recrystallization with C2–C5 alkanols were studied in this paper. Ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and 1-pentanol were used as solvents. The solubility of BOB extractives at the solvents boiling point was determined empirically. It was found that, with an increase of the boiling point of the solvents, the solubility of the extractives increased, reaching up to 486 g/L in 1pentanol at 142.2 °C. Recrystallization yields reached up to 67 wt% using 2-butanol and the purity of betulin up to 96 wt% using 2‐propanol as a solvent. Considering the yield of recrystallization, betulin content, boiling point of the solvent, solvent loss, toxicity and eco-friendliness of the solvent, ethanol was chosen to be the most suitable solvent for industrial scale purification of betulin in the BOB extractives. Using only one recrystallization step the content of betulin was increased by 20% — from 75 to 95 wt% and the yield of recrystallization was 32.1 g per liter of solvent.

State and Properties of a Carbon Sorbent Modified with a Bioactive Substance

A method for introducing small amounts of betulin (up to 1 wt %) into a mesoporous carbon material with a preset specific surface area of 300–310 m2/g, total specific pore volume of 0.35–0.40 cm3/g, and average pore diameter of 5.2–5.5 nm has been proposed and studied. The betulin content in the solid carbon matrix and in the used organic solvents has been determined by means of a gas chromatography–mass spectrometry technique. By means of scanning electron microscopy together with X-ray microanalysis and IR Fourier spectroscopy, the state of betulin on the outer and inner surfaces of carbon granules of different fractional composition, as well as in thin carbon layers, has been studied. It has been found that the main fraction of betulin is predominantly deposited on the outer surface of the globular particles of a porous carbon material. The properties of betulin impregnated into a carbon sorbent and the process of its dynamic desorption under the conditions simulating the biological environment of the gastrointestinal tract are studied.

H2S-induced NO/SNO positively promotes betulin production in Betula platyphylla

S-nitrosoglutathione reductase (GSNOR) is the pivotal enzyme that mediates cellular levels of nitric oxide (NO) and S-nitrosylation (SNO) in plants. We have previously reported that GSNOR deficiency enhanced betulin production. However, the physiological function of NO/SNO in plants remains unclear, especially for SNO. This study aimed to reveal the role of hydrogen sulfide (H2S)-induced NO/SNO on betulin production in birch (Betula platyphylla). Results showed that the H2S donor sodium hydrosulfide(NaHS)enhanced the production rates of NO, SNO and betulin in birch suspension cells to maximum levels, which was 92.59%, 50.09% and 122.58% higher than that in the control, at 24 h after 1 mmol·L−1 NaHS treatment. NaHS also promoted NO burst, SNO production and the gene expression of farnesyl pyrophosphate synthase, lupeol synthase and squalene synthetase related to betulin synthesis in birch GSNOR transgenic plants by RNA interference silencing. Similarly, pretreatment of the birch suspension cells with the GSNOR inhibitor 3-(5-(4-(1H-imidazol-1-yl) phenyl)-1-(4-carbamoyl-2-methylphenyl)- 1H-pyrrol-2-yl) propionic acid (N6022) promoted NaHS-triggered NO/SNO and betulin production, and the betulin content under NaHS and N6022 treatment was significantly higher than that under NaHS treatment or NaHS and NO specific scavenger 2-(4-carboxyphenyl)-4,4,5,5 -tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, pretreatment of the birch suspension cells with cPTIO or NO synthase inhibitor (sodium azide or NG-nitro-L-Arg methyl ester) significantly decreased NaHS-triggered NO/SNO and betulin production. Overall, the above results first verified that endogenous SNO mediated hydrogen sulfide-induced betulin production at the pharmacological and genetic levels, and the homeostasis of NO/SNO mediated hydrogen sulfide-induced betulin production. This study provides new insights into the homeostasis of NO/SNO in plant secondary metabolite production.

GSNOR deficiency enhances betulin production in Betula platyphylla

This paper showed that GSNOR mediated betulin production from genetic and pharmacological levels. The aim of this study was to investigate the relationship between S-nitrosoglutathione reductase (GSNOR) and betulin production. Treatment of birch suspension cells with 20 μmol/L GSNOR inhibitor 3-(5-(4-(1H-imidazol-1-yl) phenyl)-1-(4-carbamoyl-2-methylphenyl)-1H-pyrrol-2-yl) propio- nic acid (N6022) markedly reduced gene expression of GSNOR, and increased at least two times in betulin content and gene expression of lupeol synthase (LUS), a key enzyme in betulin biosynthesis. GSNOR transgenic plants by RNAi silencing also lowered gene expression of GSNOR and increased betulin content and gene expression of LUS. Our study also showed that S-nitrosothiols (SNO) content increased in birch suspension cells treated with N6022 and GSNOR transgenic birch plants, about three times that in the non-transgenic birch. The above results verified that GSNOR deficiency mediated betulin production from genetic and pharmacological levels, and indicated that protein S-nitrosylation mediated plant secondary metabolite production.

An approach to effective green extraction of triterpenoids from outer birch bark using ethyl acetate with extractant recycle

The outer birch bark of Betula pendula Roth. was extracted using dry ethyl acetate, wet ethyl acetate (∼3.5% H2O) by the classical reflux boiling. The yields of the extracts, the content of triterpenoids and polar impurities were compared with the results of the extractions employing aqueous ethanol (5% H2O). A higher content of betulin and a lower content of polar impurities were found for ethyl acetate extractions compared with the ethanol-based experiments. Alkaline washing of betulin concentrates was proposed as an alternative cost-effective method for purifying extracts of polar impurities. The uptake of residual miscella on the depleted birch bark was assessed to be up to 200–300% relative to the bark charge. An efficient recovery of the residual ethyl acetate by hydrodistillation from the extracted bark was demonstrated. An assessment of stepwise energy consumption for the extraction, concentration of extract, solvent recovery established that the extractions using ethyl acetate are significantly energy-saving, compared with ethanol usage. Batch extraction of birch bark employing ethyl acetate (96.5%) with hydrodistillation of residual solvent from raw materials can be used for a small-scale process to obtain betulin concentrates. According to the scheme proposed, complete recycling of the organic and aqueous phases is carried out and extractant losses are practically excluded. Considering large-scale processes, the continuous extraction scheme with solvent separate recovery from waste materials in the special equipment is feasible with dry solvent application. Extracting birch bark with “green” extractant ethyl acetate is beneficial, since it provides extracts containing less polar impurities and significantly reduces energy consumption and extractant losses comparing with use of polar extractants.

Relationship between ethanolic extracts of yellow birch and tree characteristics

The traditional wood industry has gone through an important crisis over recent years due to a decline in demand for certain traditional products coupled with a decreased demand due to competing developing economies, among others. In this context, the production of high value-added products and the exploration of new markets could promote the transition to a more resilient forest industry. One of the up and coming markets involves the plant-based extracts, which can be used in several industries, such as pharmaceuticals, cosmetics and nutraceuticals. In order to better understand the potential that biorefining products from yellow birch (Betula alleghaniensis) have to produce high value-added extracts, this study evaluated the relationship between total phenols, major triterpenes and phytosterols content in wood and bark of the studied species and selected tree characteristics. Results showed that the majority of the studied chemical compounds were related to tree health. The wood of non-vigorous trees presented the highest total phenols content among the three vigour classes, with an average of 394.9±138.6mg GAEg−1 of oven-dry extract, while presenting the lowest β-sitosterol content (3.0±0.9mgg−1). The bark of moribund trees contained the lowest total phenols content (317.6±46.8mg GAEg−1), but the highest betulin content (6.6±4.2mgg−1). Variations in bark compounds were also frequently correlated with the presence of fungal fruiting bodies on the stem, being associated with lower extract values for the betulin, lupeol and lupenone. Tree vigour and the presence of sporocarps in the stem explained a significant part of the variation in the studied chemical compounds from wood and bark. A remaining unexplained variation may be attributed to stand-level conditions.

Simple and efficient process for large scale preparation of betulonic acid from birch bark extracts

A bioactive triterpenoid betulonic acid is a valuable synthone for preparation of lupane- and oleanane-type derivatives with a wide spectrum of medical applications. A large-scale, cost-effective, and simple preparation of betulonic acid from birch (Betula pendula) bark is presented herein. The process includes four steps with the acid overall yield of 51% counting on betulin content in the extract and with the acid purity of 90% or more. The process can be employed for batch production of betulonic acid.

Endogenous and Exogenous Calcium Involved in the Betulin Production from Submerged Culture of Phellinus linteus Induced by Hydrogen Sulfide.

Using pharmacological and biochemical approaches, Ca(2+) involved in the betulin production in mycelia of Phellinus linteus induced by hydrogen sulfide (H2S) were investigated. The results showed that 2 mM H2S donor NaHS or 10 mM CaCl2 was found to enhance the betulin content in the mycelia of Phellinus to the maximum, which were 112.43 and 93.24% higher than that in the control, respectively. Further, NaHS and CaCl2 co-treatment also showed positive outcome, which were 128.95 or 24.52% higher than that in the control or NaHS treatment. At the same time, NaHS also enhanced the content of Ca(2+) and CaM. But, the above positive inductive effects for Ca(2+), CaM, and betulin production can be blocked with either Ca(2+) channel blocker (LaCl3, 2-aminoethoxydiphenyl borate) or Ca(2+) chelator (ethylenediaminetetraacetic acid (EDTA)). Among of them, betulin content was reduced 35.06% by NaHS and EGTA to the minimum, and this reduction could be reversed by the application of CaCl2 (NaHS + EGTA + CaCl2). From above results, it can be concluded that endogenous and exogenous calcium involved in the betulin production from submerged culture of P. linteus induced by hydrogen sulfide.