In a previous study, we fractionated crude polysaccharide (AME-CP) with macrophage-stimulating activity from a hot-water extract (AME) of Astragalus membranaceus. AME-CP contained glucose (Glc) as a main component sugar, suggesting that it might be rich in starch-like compounds (SLC). To enhance the immunostimulating activity of AME-CP by pruning SLC rarely known to contribute to activity, hydrolysate (AME-SH) was prepared by digesting with starch-related enzymes, including α-amylase and amyloglucosidase. AME-SH was found to contain substances with molecular weights ranging from 3.9 to 84.4 kDa. These substances were primarily composed of galactose, galacturonic acid, Glc, arabinose, rhamnose, and mannose. AME-SH significantly enhanced the production of macrophage-stimulating factors, including nitric oxide (NO), interleukin (IL)-6, and IL-12, in RAW 264.7 cells compared to AME-CP. Treatment of splenocytes isolated from C3H/HeN mice with AME-SH not only promoted IL-6 secretion, but also induced mitogenic activity. In addition, AME-SH promoted the secretion of hematopoietic growth factors including IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in Peyer's patch (PP) cells and stimulated bone marrow cell proliferation through these PP cells. In conclusion, hydrolysate (AME-SH) digested from AME-CP with starch-related enzymes could be used as a potential immunostimulant.

To utilize pepper (Piper nigrum) as an immunostimulatory agent, we isolated macrophage stimulating polysaccharides from pepper and investigated their macrophage activating activities. Hot-water extracts (HW) of black pepper (BP) and white pepper (WP) were prepared, and their macrophage stimulating activities were evaluated using RAW 264.7 cells. BP-HW significantly promoted the secretion of macrophage stimulating factors such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-12 compared to WP-HW. When BP and WP-HW were fractionated into crude polysaccharides (CP) and low molecules (LM) by ethanol precipitation, BP-CP demonstrated significantly more potent activity. Furthermore, BP-CP not only induced mRNA gene expression of macrophage activation factors, but also promoted nuclear localization of p65 and c-Jun. In addition, component sugar analysis revealed that glucan-type polysaccharides in BP-CP played a crucial role in macrophage activation. Taken together, these findings suggest that black pepper has industrial applicability not only as a spice, but also as an immunostimulatory functional material.

To increase industrial applicability of Astragalus membranaceus (AM) as immunostimulating materials, hot-water extract (AME) was prepared from AM and fermented with Kimchi-lactic acid bacteria (Lactobacillus sakei & Leuconostoc mesenteroides) to prepare fermented AM-postbiotics (FAME). Although FAME prepared from AM-postbiotics did not show a significant enhancement in macrophage stimulating activity compared to non-fermented AME, crude polysaccharide (FAME-CP) fractionated by EtOH precipitation from FAME showed significantly higher macrophage stimulating activity than AME-CP. Compared to AME-CP, FAME-CP showed dramatic changes in component sugar and molecular weight distribution. FAME-CP was a polysaccharide with a major molecular weight distribution of 113.4 kDa containing Man (44.2%), Glc (19.3%), Gal (10.2%), GalA (10.2%), and Ara (7.4%) as sugar components. FAME-CP with enhanced macrophage stimulatory activity not only increased expression levels of mRNA genes encoding macrophage-activated factors (iNOS, TNF-α, MCP-1, IL-6, and COX-2), but also led the nuclear translocation of activated p65 and c-Jun. In conclusion, crude polysaccharide from AM-postbiotics fermented with lactic acid bacteria could increase industrial applicability as a functional material with enhanced immunostimulating activity than AME-CP.

To produce an intestinal immunomodulatory beverage containing Centella asiatica extract (CAE), three types of CAE-added beverage prototypes were prepared, and their immunomodulatory activities and marker compounds were analyzed. As a result of the cytotoxicity assessment, all the beverages did not show significant toxicity compared to the control group. Next, the immunomodulatory activities of the beverage prototype were evaluated using the inflammatory model of IL-1β-induced intestinal epithelial cell line. All the samples significantly reduced the production of IL-6, IL-8, and MCP-1 in a CAE concentration-dependent manner. In addition, CAE-added beverages inhibited NO, IL-6, and IL-12 production in LPS-induced RAW 264.7 cells. When the major triterpenoids, as marker compounds for the production of CAE-added beverages, were analyzed by HPLC-DAD, only asiaticoside was detected beyond the limit of quantification, while madecassoside, madecassic acid, and asiatic acid were not detected. The amounts of asiaticoside in CAE-added beverage prototypes were confirmed in No. 1 (19.39 μg/mL), 2 (19.25 μg/mL), and 3 (19.98 μg/mL). In conclusion, the results of this study suggested that CAE-added beverage prototypes induced immunomodulatory effects in the intestinal inflammatory cell line models and asiaticoside could be used as a marker compound for CAE-added beverage production.

Centella asiatica (C. asiatica) has been widely used in food, cosmetics, and pharmaceutical industry as a functional material. In a previous study, we have investigated not only pharmacological effects such as antioxidative and anti-inflammatory effects, but also analyzed various functional ingredients. In this study, triterpenoids were analyzed using HPLC-DAD to determine marker compounds among functional ingredients. When triterpenoids were analyzed, asiaticoside from C. asiatica was determined as an optimal marker compound. Next, specificity, linearity, limited of detection (LOD), limited of quantification (LOQ), precision, accuracy, and range were evaluated using HPLC-DAD to determine asiaticoside contents in C. asiatica juice and extracts. The specificity was elucidated by chromatogram and retention time using an established analytical method. The coefficient of correlation obtained was 0.9996. LOD was 4.99 μg/mL and LOQ was 15.12 μg/mL. Intra- and inter-day precision of asiaticoside were determined to be 0.48~1.68% and 0.08~1.09%, respectively. Furthermore, the recovery rate of asiaticoside was 98.88% and the analytical range of Field-70E was determined to be 0.625~10 mg/mL. As a results of evaluating ABTS, DPPH, and FRAP antioxidative effect, Field-70E showed potent antioxidant activities. Results of this study could be used as basic data for quality standardization of C. astiatica juice and extracts.

To investigate the anti-inflammatory activity of submerged culture using Cordyceps militaris mycelium, culture-including mycelia was extracted and lyophilized into postbiotics (hot-water extract; CM-HW). HW was fractionated into crude polysaccharide (CM-CP) by ethanol precipitation, and CM-CP was further dialyzed into CM-DCP by dialysis with running water using 12~14 kDa dialysis tube. When the cytotoxicity of subfractions against cells was assessed, no subfraction had a cytotoxic impact that was substantially different from the control groups. In an inflammatory model using LPS-stimulated RAW 264.7 cells, CM-DCP significantly decreased IL-6 and MCP-1 production levels compared to the LPS-control group. CM-DCP also inhibited IL-6 and IL-8 secretion in HaCaT keratinocytes stimulated with TNF-α and IFN-γ. In the meanwhile, the neutral sugar content and mannose ratio of anti-inflammatory CM-DCP were higher than the other fractions, and CM-DCP contained β-1,3/1,6-glucan of 216.1 mg/g. High pressure size exclusion chromatography revealed that CM-DCP contained molecules with a molecular weight range of 5.6 to 144.0 kDa. In conclusion, postbiotics of C. militaris mycelium significantly promoted anti-inflammatory activity, suggesting that neutral polysaccharides including Glc and Man contribute to the anti-inflammation in RAW 264.7 or HaCaT cells.

To enhance the bioavailability and bioactivities of mixed herbal medicines (RW), they were fermented with lactic-acid bacteria isolated from kimchi into postbiotics (FRW). Then, from the results of the 16s rRNA sequencing analysis, lactic acid bacteria isolated from kimchi were identified to be of two species, namely Lactobacillus sakei and Leuconostoc mesenteroides. The FRW prepared from the RW were extracted using hot water (HW) and 70% EtOH (EtOH) for comparison of their macrophage-stimulating activities. Based on a comparison of the activities of the FRW extracts, nitric oxide (NO) production of HW was significantly higher than that in EtOH. An analysis of the chemical properties of the extracts showed that HW had higher contents of neutral sugar and uronic acid than EtOH as well as contained a large amount of glucose. In addition, crude polysaccharide (CP) was prepared to enhance the macrophage-stimulating activity. The FRW-CP not only secreted immunostimulatory mediators but also increased the expression of immunostimulatory genes (iNOS, TNF-α, MCP-1, and IL-6). The fractionated FRW-CP contained about 90% neutral sugars, and these sugars were mainly composed of glucose, galacturonic acid, and arabinose. Thus, FRW prepared by fermentation of RW with kimchi lactic acid bacteria were found to be immunostimulatory modulators.

To confirm the applicability of crab apple, the composition and content of amino acids and minerals with ‘Fuji’ apple were measured. Apple and crab apple were extracted with water, 70% and 100% ethanol, respectively. The contents of polyphenols, flavonoids, and ascorbic acid, antioxidant activities of these extracts were measured. The amino acid composition of apples comprised a total of 17 amino acids. The total amino acid contents of apple and crab apple were 2,050.45 mg/kg and 900.05 mg/kg, respectively. For minerals, the total mineral content of apple and crab apple were 489.14 mg%, 529.77 mg%, respectively. The contents of polyphenols, flavonoids, and ascorbic acid of apple and crab apple extracts were highest in 70% ethanol extracts. The content of polyphenols, the crab apple extract, showed a generally higher content than the apple extract. The content of flavonoids, apple and crab apple extracts revealed no difference between extracts. The content of ascorbic acid, apple extract showed a generally higher content than the crab apple extract, but there was no significant difference between extracts. In the case of DPPH and ABTS radical scavenging abilities, 70% ethanol extract showed the highest antioxidant activity, and crab apple showed higher activity than apple extracts.

To improve the shelf-life of Centella asiatica, Centella asiatica was treated with gel packs containing slow-released chlorine dioxide (ClO2) gas at 3-5 ppm for 20 days at 4℃. The weight loss rate, as well as the changes in pH, color, and texture of the treated samples, were investigated. The weight of the control and ClO2 gas-treated samples was decreased during the storage period. The change in weight of the control was slightly faster than that of the samples treated with 3 and 4 ppm ClO2 gas. The pH of the control and the ClO2 gas treated samples were decreased during the storage period and there was no significant difference between the control and ClO2 gas treated samples. Concerning color (lightness, redness, and yellowness) changes of Centella asiatica during the storage period, there was no significant difference between the control and ClO2 gas treated samples. The change in shear force in the leaf and stem of Centella asiatica during the storage period was slightly lower in the 4 ppm ClO2 gas treated samples (in the leaf) compared to the control and 3 and 4 ppm ClO2 gas treated samples (in the stem) compared to the control and 5 ppm ClO2 gas treated sample.

The purpose of this study was to assess consumption and satisfaction with fresh-cut produce in Korean adults. In this study, an online survey November 9~13, 2020 was conducted with 982 Korean adults age 19~64 (490 males and 492 females) with experience in purchasing fresh-cut produce in the past year. Among all respondents, the percentage with experience in buying ‘washed or peeled vegetables’ was the highest (71.18%), followed by those in buying ‘packaged salads/sprouts/ssam vegetables’ (64.15%), ‘pre-cut fruits/fruit cups’ (59.98%), ‘pre-cut or minced vegetables’ (56.01%), and ‘washed or peeled fruits’ (53.67%), respectively. Among the types of fresh-cut produce, ‘packaged salads/sprouts/ssam vegetables’ showed the highest satisfaction score at 3.73 pts, and the overall satisfaction score of fresh-cut produce consumption was also 3.73 pts. Compared to male respondents, female respondents showed a significantly higher percentage of respondents that purchased ‘packaged salads/sprouts/ssam vegetables’ (p=0.0409), significantly higher satisfaction scores (p=0.0124), and a higher percentage of respondents considering ‘freshness’ important when purchasing (p<0.0001). Additionally, the group with high household income comprised a significantly higher ratio of respondents considering ‘eco-friendly or organic status’ important. The study results may facilitate identify areas for improvement of fresh-cut produce in the future.

Maca roots (Lepidium meyenii) are an important medicinal herb that have long been used by the Andes-indigenous peoples and South Americans. In Korea, recently, it has attracted attention as a health food material because of nutritional values and physiological activities. The purpose of this study was to investigate the industrial applicability of maca (red and golden varieties; R&G) as immunostimulating materials. In the macrophage stimulating assay using RAW 264.7 cells at 125~500 μg/mL of non-cytotoxicity doses, G-HW showed the most potent production of TNF-α, IL-6 and nitric oxide compared to red maca or the other extracts. The general component analysis results showed that all extracts comprised more than 90% neutral sugars with small amounts of uronic acid and protein. Meanwhile, component sugar analysis showed the difference in the content of uronic acids of cold- and hot-water extract. Additionally, the further fractionation of G-HW into crude polysaccharide (G-CP) greatly enhanced the macrophage stimulating activity, and G-CP contained macromolecules over 144 kDa, comprised mainly of glucose and galacturonic acid (51.0 and 34.9%). In conclusion, crude polysaccharide from maca showed industrial applicability as immunostimulating material, and especially golden maca showed higher macrophage stimulating activity than red maca.

Compound K (20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol)는 진세노사이드의 활 성성분이다. Compound K는 경구 투여 후 Rb1, Rb2 및 Rc가 사람의 장내 미생물의 β-glucosidase에 의 해 생물전환 과정을 거쳐 생성된다고 알려져 있다. 본 연구는 생물전환된 인삼농축액에서 얻은 compound K를 이용해 항염증 및 독성을 조사하였다. 세포독성평가 결과, compound K는 0.001∼1 μg/mL의 농도 범위에서 유의적인 세포독성은 나타나지 않았으며, LPS로 염증이 유발된 RAW 264.7 세포에서 TNF-α, MCP-1, IL-6 및 NO의 생성을 억제하는 것으로 확인되었다. 동일 농도범위에서 TNF-α 및 IFN-γ로 염증이 유발된 HaCaT 세포는 compound K의 처리로 인해 IL-8의 생성을 감소시키는 것으로 나타났지만, IL-6의 경우 일부 농도에서 생성을 감소시켰으나, 통계적인 유의성은 나타나지 않았다. Brine shrimp를 이 용한 치사율 검정법에서 compound K의 LC50는 0.37mg/mL로 다소의 독성을 함유하고 있는 것으로 나타 났으나 compound K가 35% 고함유된 생물전환물은 LC50가 0.87mg/mL로 나타나 상대적으로 낮은 독성 을 보였다. 따라서 이 생성물은 향후 여드름 완화용 화장품 개발에 사용할 수 있는 매우 우수한 기능성 소 재가 될 수 있을 것으로 기대된다.

To increase the utilization of Centella asiatica (CA), enzymes such as cellulase and pectinase were added and the physicochemical properties of the treated CA were analyzed. In addition, apple-CA jam was prepared using the enzyme-treated CA, which had the best antioxidant properties, and the physicochemical and sensory qualities of the jam were measured. There was a high content of ascorbic acid, polyphenols, flavonoids, reducing sugar, amino acid, minerals and DPPH radical scavenging activity in the enzyme-treated group. The antioxidant component and activity in the jam prepared by adding enzyme-treated CA increased with an increase in the amount of enzyme-treated CA. In the soluble solids, the higher the amount of enzyme-treated CA, the higher the value, but there was no significant difference in pH. The sensory evaluation of the jam, in particular the taste, showed that the highest preference was observed when the enzyme-treated CA was added in the range of 5.0~6.7%, and the control group showed the lowest preference. There was no significant difference in flavor and spreadability among the treatment groups, however, the control group showed the highest color preference. In the overall acceptability, when 5.0% of enzyme-treated CA was added, the highest acceptability was shown.

진세노사이드 Compound K는 트라이터펜계 사포닌으로써 인삼의 잎, 줄기, 뿌리등에서 발견된다. 본 연구는 효소 Plantase를 이용하여 인삼 추출물로부터 고부가가치의 진세노사이드인 Compound K를 생산하는 연구를 하였다. Plantase는 인삼추출물에서 Compound K를 매우 효율적으로 생산함을 보여 주었다. 또한 다양한 온도와 pH에서 Compound K 생산에 대한 최적의 반응을 조사한 결과 pH 5, 50 ℃에서 가장 높은 효율을 보였다. 최적 조건에서 Compound K는 전체 추출물의 35%이상 농축될 수 있음을 확인 하였다. 생물전환된 Compound K 농축물의 항균효과를 검정한 결과 여드름균인 Cutibacterium acnes KCTC 3314에 선택적인 활성을 보였다. Compound K (35% 함유) 인삼 생물전환물의 C. acnes KCTC 3314 균주에 대한 최소저해농도 측정 결과 31.25ug/mL로 확인되었다. 따라서 향후 여드름균 완화용 화장품의 잠재적 소재로 사용될 수 있을 것으로 기대된다.

To investigate the industrial availability of liquid fermentation (PL-ferment) by Phellinus linteus mycelium as a postbiotics for the inhibition of inflammation, PL-ferment was fractionated into culture supernatant (CS), hot-water extract (HW) from PL-ferment, EtOH-precipitate (CP) fractionated from HW, and the dialysate (DCP) of CP. Compared to the other fractions, DCP which is expected to contain exopolysaccharide (EPS) as the major component, significantly decreased the production of NO, IL-6, and MCP-1 in LPS-induced RAW 264.7 cells, and IL-6 and IL-8 in TNF-α and IFN-γ-induced HaCaT cells. The general component analysis results showed that no significant difference in components was observed between the fractions, whereas sugar composition analysis revealed that DCP had decreased glucose and increased mannose contents compared to the other fractions. This suggests that mannose played an important role in the anti-inflammatory activity of the active fraction, DCP. Molecular weight distribution analysis revealed that DCP was mainly composed of low-molecular-weight material-removed high-molecular-weight polysaccharides of 18–638 kDa, suggesting that EPS originated from P. linteus EPS. In conclusion, our results suggest that the DCP of P. linteus mycelium fermentation using the anti-inflammatory activity could be used industrially as postbiotic material.

To utilize Malus pruniforia Borkh. as a functional material, cold-water (CW), hot-water (HW), and 70% ethanol (EtOH) extracts were prepared, and their antioxidant and anti-inflammatory activities were compared. The antioxidant activity of the HW extract evaluated by ABTS and DPPH radical scavenging and FRAP activity was significantly effective. The total polyphenol content of the HW extract was also higher by 15.5±0.7 mg GAE/g extract compared to other extracts. The EtOH extract showed significantly decreased TNF-α (39.8%), IL-6 (65.5%), and NO (34.9%) levels in RAW 264.7 cells compared to the LPS-induced control group. The levels of IL-6 (21.1%) and IL-8 (19.3%) were significantly decreased by treatment of EtOH extract in HaCaT keratinocytes induced with TNF-α and IFN-γ. The UHPLC-MS results indicated that the EtOH extract might have chlorogenic acid and phlorizin as the major compounds. This was validated using HPLC-DAD, which showed that the EtOH extract had higher levels of chlorogenic acid and phlorizin (1,185±58 and 470±10 μg/g extract, respectively). In conclusion, the present study suggested that the anti-inflammatory activity of the EtOH extract was more effective than the CW and HW extracts, and chlorogenic acid and phlorizin could be used as indicator compounds and functional substances.

본 연구는 병풀(Centella asiatica) 두 품종(‘자이언트 타이 거 케어’, ‘굿병풀’)의 생장 특성과 추출용매에 따른 항산화 활성 및 Triterpenoid 함량을 조사하기 위해 수행되었다. ‘자이언트 타이거 케어’와 ‘굿병풀’의 정식 후 생장 특성은 정식 41일 후 엽과 엽병의 길이 생장에서 ‘굿병풀’이 유의적으로 높았다. 하지만 이후 수확기(정식 104일 후)까지 생장속도는 유사하였다. 정식 후 104일에 수확한 ‘자이언트 타이거 케어’의 식물체 크기와 관련된 생장(엽면적, 엽병두께, 엽병 길이, 생체중)이 ‘굿병풀’보다 유의적으로 높았다. 냉수, 열수, 50% 에탄 올, 70% 에탄올 추출물의 자유라디컬 소거능과 환원능으로 평가된 항산화 활성과 총 페놀 함량은 두 품종에서 모두 70% 에탄올 추출물에서 높았다. 추출 용매별 Triterpenoid 함량을 분석한 결과, 두 품종의 주요 Triterpenoid는 Madecasoside 와 Asiaticoside로 확인되었다. 품종별 총 Triterpenoids 함량은 ‘자이언트 타이거 케어’의 50% 에탄올 추출물과 ‘굿병풀’ 의 70% 에탄올 추출물에서 가장 높았으며, ‘굿병풀’이 ‘자이언트 타이거 케어’보다 높았다. 하지만 정식 104일 후 수확량 은 ‘자이언트 타이거 케어’가 ‘굿병풀’보다 3배 많아, 동일한 재배 면적(3.3m2 ) 에서 생산 가능한 총 Triterpenoids 함량은 ‘자이언트 타이거 케어’가 2.459mg으로 ‘굿병풀’(1.103mg) 보다 약 2.2배 높았다. 따라서 항산화 활성이 가장 높고 단위면 적당 총 Triterpenoids 생산량이 높은 ‘자이언트 타이거 케어’ 를 재배하는 것이 경제적일 것으로 생각된다.

Centella asiatica (CA) is a vegetable that has been used for medicinal purposes for a long time, but it is not well known in Korea. In this study, the approximate analysis, physicochemical properties, free sugars, free amino acids and minerals of native and improved CA cultivated in Chungju area were measured. And the antioxidant contents and activities of CA solvent extracts were measured. There was no significant difference between native and improved species. As for the characteristics of CA, it was confirmed that the improved species had a large weight, size and a tough texture. Glucose was detected in native CA, fructose, glucose and sucrose were detected in improved species. A total of 15 free amino acids were detected and the content was slightly higher in improved species than in native species. The mineral content in CA was slightly higher in the improved species, and the detected minerals were Na, P, Ca, K, Mg and Fe. Polyphenols, ascorbic acid and flavonoids in each extraction solvent (water, 70% and 100% ethanol) were all higher in the improved species than in native species. And it was found that the antioxidant activities of ethanol extracts were higher than that of water.

Centella asiatica (CA) has been used as a nutritional plant as well as a traditional herbal medicine around the world. This study, quality component (proximate composition, total polyphenol, and triterpenoid compound), and antioxidant and anti-inflammatory activities of CA dried using various methods were investigated. Proximate compositions of CA with different drying methods included a large amount of carbohydrates, crude protein, crude ash, and crude fiber. Among the drying methods, cold drying provided the highest total polyphenol content and antioxidant activity, while hot-air drying at 75℃ provided the lowest total polyphenol content and antioxidant activity. In addition, when the major triterpenoid compounds of CA were analyzed, the highest content of asiaticoside of triterpenoid glycoside was obtained with all drying methods. With respect to the total triterpenoid, the highest content was obtained with cold drying (68.8 mg/g) whereas natural drying (31.4 mg/g) provided the lowest content. In anti-inflammatory activity of LPS-stimulated RAW 264.7 cells, EtOH extract of cold drying showed a significantly higher inhibitory activity in comparison to the other drying methods. In conclusion, it is considered that the cold drying method is suitable for industrial preparation of functional materials with high physiological ingredients, and antioxidant and anti-inflammatory activities from CA.

After ethanol (BM-E and RW-E) and hot-water (BM-HW and RW-HW) extracts were fractionated from two herbal mixtures (BM and RW), their physiological activities were investigated. All extracts consisted of more than 50% of neutral sugar, with their total polyphenol levels higher than flavonoid levels. Radical scavenging activities of EtOH extracts remained significantly higher compared to that of hot-water extracts, and in particular, RW-E showed consistently higher antioxidant activity than BM-E. When anti-inflammatory activities of the extracts were evaluated by LPS-stimulated RAW 264.7 cells at 10~500 μg/mL non-cytotoxicity doses, BM-E showed significantly higher levels of TNF-α, IL-1β, IL-6, and nitric oxide inhibitory activity than those of hot-water extracts and RW-E. Murine peritoneal macrophage cells were shown to be enhanced in crude polysaccharides (BM-CP and RW-CP fractionated from BM-HW and RW-HW) compared to hot-water extracts and polysaccharide K (PSK, positive control). Especially, RW-CP exhibited higher activity than BM-CP, and component sugar analysis showed that BM-CP mainly contained galacturonic acid, glucose, arabinose, galactose, and xylose (34.5%, 33.9%, 16.1%, 7.1%, and 6.3%, respectively), whereas RW-CP showed different measurements (29.5%, 59.2%, 5.0%, 4.5%, and 0.2%). In conclusion, two herbal mixtures could contain varying sets of physiological activities dependent on different extraction and fractionation methods.