Advances in biosynthesis of galanthamine and huperzine A / 中草药

Acetylcholinesterase inhibitors (AChEIs) isolated from plants have become the mainstream of clinical treatment for Alzheimer’s disease because of their high efficiency and low toxicity. At present, the production of galantamine and huperzine A still mainly rely on plant extraction since they are not chemically synthesized on a large scale in industry. However, with the sharp rise of social demand, the contradiction between supply and demand has become increasingly prominent due to the difficulty in cultivation and the poor abundance of effective substances. Developing new alternative resources and taking the advantage of metabolic engineering for the production of AChEIs such as galantamine and huperzine are the efficient ways to alleviate the current contradiction. Here, the current development status of alternative resources was summarized and the progress of biosynthesis of galantamine and huperzine A during the past few years was reviewed.

Efficacy and safety of huperzine A in treating patients with mild cognitive impairment: a systematic review and Meta-analysis / 中国中药杂志

To evaluate the efficacy and safety of huperzine in treating patients with mild cognitive impairment. The randomized controlled trials(RCT) were retrieved from EMbase, Cochrane Library, PubMed, CNKI, Wanfang and VIP. The methodology quality of the included studies was evaluated, and a Meta-analysis was performed using RevMan 5.3 software. A total of nine RCTs were included. The Meta-analysis results showed that compared with placebo, Huperzine significantly increased the scores of memory quotient(MQ) and mini-mental state examination(MMSE). However, there was no statistical difference between oral tablet and capsule. Compared with placebo, huperzine A was superior in the scores of MQ and MMSE. Huperzine is safe with mild side effects. Due to the low quality of original studies, more high-quality studies are needed to verify its efficacy.

TMT proteomics and bioinformatics to analyze protein differences in different Hup A content of thallus of Huperzia serrata / 中草药

Objective: To analyze the differences in protein of thallus of Huperzia serrata (in vitro cultures, strains A, B, and C, respectively) with different Hup A content, and to explore related enzymes that may be synthesized with Hup A accumulation. Methods: Quantitative proteomics was performed in vitro cultures of Huperzia serrata with different Hup A content using quantitative proteomics tandem mass tag (TMT) techniques, followed by differential protein analysis: GO, KEGG and other biological information analysis. Results: Strain B hadthe lowest Hup A content and strain C had the highest Hup A content, which was twice than that of B. There were 78 differential proteins between the strain B and C. Analysis of differential protein GO enrichment showed that MF accounted for 28.75%; Analysis of differential protein expression showed that three strains shared two differential proteins (P93541, Q8RXU4) in the alkaloid metabolic pathway starting from amino acids. P93541 protein was down-regulated in the low-yield strain B and up-regulated in the relatively high-yield strains A and C. The Q8RXU4 protein was up-regulated in the low-yield strain B and down-regulated in the relatively high-yield strains A and C. Conclusion: This study found that the difference in Hup A content was positively correlated with the protein expression. Two enzymes P93541 and Q8RXU4 that may be related to Hup A accumulation were analyzed, providing a basis for bioinformatics analysis of Hup A biosynthesis.

Preparation and Pharmacokinetics of Huperzine A Micro-porous Osmotic Pump Pellets / 中国药学杂志

OBJECTIVE: To prepare huperzine A micro-porous osmotic pump pellets and to investigate the pharmacokinetic properties in Beagle dogs. METHODS: The extrusion-spheronisation method was used to prepare the core of huperzine A pellets which then coated by fluid-bed coating technology. Central composite design-response surface method was used to optimize the prescription of coating layer.Then Zero-order, First-order and Higuchi equation of cumulative release with time were fitted to study its release characteristics.The commercially available huperzine A tablets were used as reference preparations to investigate the in vivo pharmacokinetics of huperzine A micro-porous osmotic pump pellets, and the bioequivalence of the two preparations were compared. RESULTS: The formula of coating was optimized as followsEC of 61.5%, PEG400 of 10.5%. Zero-order kinetics existed in the release of the pellets in 24 h. Moreover, the osmotic pressure-controlled delivery was greatly responsible for drug release. In vivo study showed that tmax and ρmax of huperzine A micro-porous osmotic pump pellets were significantly lower than that of the reference preparation, and its t1/2 was significantly prolonged compared with the reference preparation, the relative bioavailability was 95.8%. CONCLUSION: Huperzine A micro-porous osmotic pump pellets had a better sustained release effect in the Beagle dog and have a good correlation in vivo.

Study on preparation of immobilized acetylcholinesterase microreactor and its application in CMM inhibitors screening / 中草药

Objective To prepare an immobilized acetylcholinesterase (AChE) microreactor and establish a rapid screening method for Chinese materia medica (CMM) acetylcholinesterase inhibitors (AChEIs). Methods A novel immobilized AchE microreactor was prepared by crosslinking with glutaraldehyde, using aminated magnetic microspheres as carrier. The characterizations were conducted by physicochemical properties and chromatographic performance. The immobilized AChE reactor was used to screen AChEIs from the Huperzia Serratum extracts. Results In the enzyme reaction system, the optimum substrate concentration was 50 μmol/L, and the incubation time was 5 min, respectively. IR characterization, specificity verification, enzyme kinetics, and stability study results all demonstrated the effectiveness of the enzyme reactor. The CMM AChEI, huperzine A, was obtained from the screening of H. Serratum extracts. Conclusion A high throughput screening method for AChEIs is established in this paper, which will be further applied and popularized.

Simultaneous determination of huperzine A and huperzine B in Seventeen species from Huperzioideae subfamily by HPLC / 中成药

AIM To establish an HPLC method for the simultaneous content determination of huperzine A and huperzine B from seventeen species Huperzioideae subfamily (including 2 ineditus new species).METHODS The analysises of tartaricacid extracts of huperzine A and huperzine B were performed on a room temperature column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of methanol-spiritus mindereri flowing at 1.0 mL/min in a isocratic elution manner,and the detection wavelength was set at 310 nm.The contents were determined by external standards.RESULTS Huperzine A and huperzine B showed good linear relationships within 0.002 0-0.30 μg,0.001 8-0.27 μg (r=0.999 9),whose average recoveries were 103.86％,101.3％ with the RSDs of 1.85％,1.30％,respectively.Huperzine A and huperzine B were found in seventeen species,and there were significant differences in contents from species to species.The content of huperzine A in Phlegmariurus hamiltonii was the highest,which reached up to 0.22％,with higher levels of it in Phlegmariurus cryptomerianus,Phlegmariurus petiolatus,and Phlegmariurus phlegmaria;The content of huperzine B was the highest in P.phlegmaria.CONCLUSION This accurate,stable and reproducible method can be used for the quality control of huperzine A and B from Huperzioideae subfamily.

Detection of Huperzine A and Huperzine B in fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate by HPLC / 生物工程学报

In this study, we established a rapid and efficient HPLC method to determine the accumulation of Huperzine A and Huperzine B in the fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate. The chloroform extracts of fermentation broth were dissolved in methanol and filtered before injection for HPLC analysis. The analysis was performed on an Agilent Eclipse plus-C18 column (250 mm×4.6 mm, 5 μm) by isocratic elution. The mobile phase was 0.015 mol/L ammonium acetate-methanol (70:30, V/V), the flow rate was 1 mL/min and the detection wavelength was set at 308 nm. Huperzine A and Huperzine B could be well separated within 25 min. Good linearity of Huperzine A was found in the range of 1.50-48.00 μg/mL (r=0.999 5), and that of huperzine B was in 0.25-7.50 μg/mL (r=0.999 7). The average recoveries of Huperzine A and Huperzine B were 106.83% and 108.06%, respectively (RSD=3.34%, 3.60%). The results demonstrate that this method can detect the content of huperzine A and huperzine B in fermentation broth simply, rapidly, accurately and in good reproducibility. Under the optimized conditions, the accumulated content of huperzine A and huperzine B were measured from the sixth to the fifteenth day. Huperzine A and Huperzine B reached the highest (12.417 0 μg/mL and 4.660 3 μg/mL, respectively) at the fourteenth and eighth days. The analysis methodology could contribute to the future study of huperzine A and huperzine B biosynthesis in C. gloeosporioides, consequently facilitate the development of new drug resources.

Improvement Effects of Citalopram Combined with Huperzine A in Aged Depression Model Rats / 中国药房

OBJECTIVE:To study the improvement effects of citalopram combined with huperzine A in aged depression model rats. METHODS:Aged rats were randomly divided into blank control group,model group,huperzine A group(0.3 mg/kg),citalo-pram group(5 mg/kg),and combination group(huperzine A 0.3 mg/kg+citalopram 5 mg/kg),10 in each group. Except for blank control group,rats in other groups received chronic unpredictable mild stress to reduce depression model. After modeling,rats were intragastrically administrated relevant drugs once a day,for 2 weeks. The depression,learning and memory behavior changes of rats in each group were observed by using open-field test,sucrose consumption test,tail suspension test,forced swimming test and Morris water maze test. RESULTS:Compared with blank control group,the horizontal crossing number,uprightness number,su-crose preference rate,crossing number in platform,percentages of target quadrant distance and time of rats in model group were ob-viously decreased (P<0.05 or P<0.01);immobility time of tail suspension and swimming,escape latency were obviously pro-longed(P<0.05 or P<0.01). Compared with model group,the depression-related indexes of rats in citalopram group and combina-tion group were obviously improved(P<0.05 or P<0.01),and combination group had better effects;the learning and memory-re-lated indexes in combination group were obviously improved(P<0.05 or P<0.01),only crossing time in platform in huperzine A group and citalopram group were obviously increased (P<0.05 or P<0.01),and other learning and memory-related indexes had no obvious changes(P>0.05). CONCLUSIONS:Citalopram combined with huperzine A can obviously improve the depression be-havior,learning and memory ability of aged rats with depression,showing better effects than citalopram alone.

Improvement Effects of Citalopram Combined with Huperzine A in Aged Depression Model Rats / 中国药房

OBJECTIVE:To study the improvement effects of citalopram combined with huperzine A in aged depression model rats. METHODS:Aged rats were randomly divided into blank control group,model group,huperzine A group(0.3 mg/kg),citalo-pram group(5 mg/kg),and combination group(huperzine A 0.3 mg/kg+citalopram 5 mg/kg),10 in each group. Except for blank control group,rats in other groups received chronic unpredictable mild stress to reduce depression model. After modeling,rats were intragastrically administrated relevant drugs once a day,for 2 weeks. The depression,learning and memory behavior changes of rats in each group were observed by using open-field test,sucrose consumption test,tail suspension test,forced swimming test and Morris water maze test. RESULTS:Compared with blank control group,the horizontal crossing number,uprightness number,su-crose preference rate,crossing number in platform,percentages of target quadrant distance and time of rats in model group were ob-viously decreased (P<0.05 or P<0.01);immobility time of tail suspension and swimming,escape latency were obviously pro-longed(P<0.05 or P<0.01). Compared with model group,the depression-related indexes of rats in citalopram group and combina-tion group were obviously improved(P<0.05 or P<0.01),and combination group had better effects;the learning and memory-re-lated indexes in combination group were obviously improved(P<0.05 or P<0.01),only crossing time in platform in huperzine A group and citalopram group were obviously increased (P<0.05 or P<0.01),and other learning and memory-related indexes had no obvious changes(P>0.05). CONCLUSIONS:Citalopram combined with huperzine A can obviously improve the depression be-havior,learning and memory ability of aged rats with depression,showing better effects than citalopram alone.

Pharmacokinetics and Tolerability of Oral Dosage Forms of Huperzine A in Healthy Chinese Male Volunteers: a Randomized,Single Dose, Three-period, Six-sequence Crossover Study / 华中科技大学学报（医学）（英德文版）

Huperzine A is a potent,reversible,and blood-brain barrier permeable acetylcholinesterase irhibitor.The aim of this study was to compare the pharmacokinetics,tolerability,and bioavailability of two formulations with the established reference formulation of huperzine A in a fasting,healthy Chinese male population.This was a randomized,single-dose,3-period,6-sequence crossover study.The plasma concentrations of huperzine A were determined by liquid chromatography tandem mass spectrometry.Tolerability was assessed based on subject interview,vital sign monitoring,physical examination,and routine blood and urine tests.The mean (SD) pharmacokinetic parameters of the reference drug were Cmax,1.550 (0.528) ng/mL;t1/2,12.092 (1.898) h;AUC0-72h,17.550 (3.794) ng.h/mL.Those of the test formulation A and test formulation B were Cmax,1.412 (0.467),1.521 (0.608) ng/mL;t1/2,12.073 (2.068),12.271 (1.678) h;AUC0-72h,15.286 (3.434) ng.h/mL,15.673 (3.586) ng.h/mL.The 90％ confidence intervals for the AUC0-72h and Cmax were between 0.80 and 1.25.No adverse events were reported by the subjects or found with results of clinical laboratory test.The test and reference products met the regulatory criteria for bioequivalence in these fasting,healthy Chinese male volunteers.All three formulations appeared to be well tolerated.

Self-microemulsifying drug delivery system for improving the bioavailability of huperzine A by lymphatic uptake

Huperzine A (Hup-A) is a poorly water-soluble drug with low oral bioavailability. A self-microemulsifying drug delivery system (SMEDDS) was used to enhance the oral bioavailability and lymphatic uptake and transport of Hup-A. A single-pass intestinal perfusion (SPIP) technique and a chylomicron flow-blocking approach were used to study its intestinal absorption, mesenteric lymph node distribution and intestinal lymphatic uptake. The value of the area under the plasma concentration-time curve (AUC) of Hup-A SMEDDS was significantly higher than that of a Hup-A suspension (<0.01). The absorption rate constant () and the apparent permeability coefficient () for Hup-A in different parts of the intestine suggested a passive transport mechanism, and the values ofandof Hup-A SMEDDS in the ileum were much higher than those in other intestinal segments. The determination of Hup-A concentration in mesenteric lymph nodes can be used to explain the intestinal lymphatic absorption of Hup-A SMEDDS. For Hup-A SMEDDS, the values of AUC and maximum plasma concentration () of the blocking model were significantly lower than those of the control model (<0.05). The proportion of lymphatic transport of Hup-A SMEDDS and Hup-A suspension were about 40% and 5%, respectively, suggesting that SMEDDS can significantly improve the intestinal lymphatic uptake and transport of Hup-A.

Detection and identification of high producing huperzine A by an endophytic fungus from Huperzia serrata / 国际药学研究杂志

Objective To screen endophytic fungal strains isolated from wild Huperzia serrata which can highly produce huperzine A. Methods The strain producing huperzine A was identified through thin layer chromatography (TLC) and high- performance liquid chromatography(HPLC) with authentic huperzine A. The fungus was identified according to its morphological characteristics and nuclear ribosomal DNA ITS sequence. Results The strain was identified as Fusarium oxysporum NSG-1 according to its morphological characteristics and nuclear ribosomal DNA ITS sequence. The amount of huperzine A produced by the fermentation liquor of this endophytic fungus was quantified to be 1.11 mg/100 ml by HPLC, which was higher than that of previously reported endophytic fungi. Conclusion Endophytic fungus producing high huperzine A can be obtained from H. serrata. The production of huperzine A based on mutagenesis and transformation of the obtained strain may adapt to the industry production.

Conditional optimization and kinetic research on producing huperzine A using Huperzia serrata in vitro / 中草药

Objective: To investigate the influence factors and dynamic varitation rule for the accumulation of huperzine A (Hup A) from thallus of Huperzia serrata, which could produce Hup A. Methods: The optimal concentration of plant growth substances for the growth of thallus of H. serrata were screened using orthogonal design method. The content of target extract from thallus of H. serrata were tested using HPLC. The kinetic curve of thallus growth was described using Logistic growth model. Results: The thallus of H. serrata could produce the most Hup A (72.45 μg/L) when cultured in 1/4 MS solid medium complemented with 12 h/d white light and the relative growth rate could reach 3 174.5%. According to the growth curve of thallus of H. serrata like "S" shape, the best successive generation date of callus culture of H. serrata was 50 d. The concentration of Hup A increased obviously after 60 d when the thallus reached the stable period and the maximal value of Hup A was 7.453 μg/g at 85 d, which meant that the rapid growth of thallus was priority to the accumulation of Hup A. Conclusion: The relational model between the accumulation of Hup A from thallus and its growth is non-growth conjugation. In addition, the growth law of thallus is consistent with Logistic growth model and the maximum specific growth rate (μmax) was 0.071 d-1.

Detection and identification of high producing huperzine A by an endophytic fungus from Huperzia serrata / 国际药学研究杂志

Objective To screen endophytic fungal strains isolated from wild Huperzia serrata which can highly produce hu?perzine A. Methods The strain producing huperzine A was identified through thin layer chromatography(TLC)and high-perfor?mance liquid chromatography(HPLC)with authentic huperzine A. The fungus was identified according to its morphological character?istics and nuclear ribosomal DNA ITS sequence. Results The strain was identified as Fusarium oxysporum NSG-1 according to its morphological characteristics and nuclear ribosomal DNA ITS sequence. The amount of huperzine A produced by the fermentation li?quor of this endophytic fungus was quantified to be 1.11 mg/100 ml by HPLC,which was higher than that of previously reported endo?phytic fungi. Conclusion Endophytic fungus producing high huperzine A can be obtained from H. serrata. The production of huper?zine A based on mutagenesis and transformation of the obtained strain may adapt to the industry production.

Pharmacological research of natural products in the treatment of Alzheimer’ s disease / 中国药理学通报

Alzheimer’ s disease ( AD) , a central nervous system degenerative disease, is characterized by progressive cognitive impairment and memory damage. Although intensive research leads to a better understanding of AD pathology, no new medi-cines are found to prevent, delay or stop the progression of AD. Three cholinesterase inhibitors ( donepezil, rivastigmine and ga-lantamine) and an N-methyl D-aspartase NMDA receptor antag-onist, memantine have been approved in clinic for AD treat-ment, but these treatments only have modest symptomatic effects for relatively short time periods. Therefore, to find effective medicines to prevent, improve and treat AD is urgent. It is well known, the traditional Chinese medicine resources are abundant in China. Chinese medicines, including the active ingredients and compounds, have been used in dementia treatment for a long history. The review is aimed to summarize the application and related mechanisms of natural products such as huperzine A, ginsenosides, L-3-n-butylphathlide, TSG and so on in AD treat-ment.

Identification of alkaloids and huperzine A-producing endophytic fungi isolated from wild Huperzia serrata / 国际药学研究杂志

Objective To screen endophytic fungal strains isolated from wild Huperzia serrata which can produce alkaloids and huperzine A (HupA). Methods Thirty-three endophytic fungal strains were obtained from H.serrata.Alkaloid production was assayed with alkaloid precipitation and acid dye colorimetry. Then, the crude extracts of alkaloids produced from fungal strains were analyzed by modulation of acetylcholinesterase(AChE) activity, TLC and HPLC. Identification of endophytic fungi producing HupA was based on morphology analysis. Results Seven alkaloids produced by endophytic fungal strains were determined based on alkaloid precipitation and acid dye colorimetry, among which strain V generated HupA in vitro. Morphology analysis showed that strain V belonged to Penicillium. Conclusion A high percentage of fungal strains isolated from H.serrata leaves has alkaloid producing potentials. It is the first report that Penicillium species produced HupA. The screening of endophytic fungi producing HupA based on alkaloid precipitation and acid dye colorimetry, AChE activity and structure identification are more efficient and directive.

Pharmacokinetics of compound huperzine a and ginkgolide b solid lipid nanospheres (SLN) after oral administration to beagle dogs / 中国药学杂志

OBJECTIVE: To investigate the pharmacokinetics of solid lipid nanoparticles loaded with huperzine A and ginkgolide B in beagle dogs, and compare the pharmacokinetics and bioavailability with huperzine A commercial tablets. METHODS: The analytes and internal standard (diazepam and glibenclamide) were extracted from plasma by liquid-liquid extraction and analyzed on a C18 column. The mobile phase consisted of acetonitrile-methanol-10 mmol · L-1 ammonium acetate (30:40:30, V/V/V, pH adjusted to 6-7 with formic acid) at a flow rate of 0.3 mL · min-1. The column temperature was 25℃. Electrospray ionization (ESI) source was applied. Huperzine A and diazepam were operated in positive mode. Ginkgolide B and glibenclamide were operated in negative mode. The precursor-product ion combinations of m/z 243.2→210.2, m/z 285.2→193.2, m/z 423.1→367.3, m/z 492.3→369.1 were used to quantify huperzine A, diazepam, ginkgolide B, and glibenclamide, respectively. Pharmacokinetic parameters were calculated by DAS2.0, and statistical analysis was done by SPSS19.0 software. RESULTS The main pharmacokinetic parameters of huperzine A in HA-GB-SLN and huperzine A commercial tablets were as follows: t1/2 (4.59 ± 1.04) and (12.02 ± 0.98) h, ρmax(5.55 ± 0.97) and (2.04 ± 0.23) ng · mL-1, tmax(2.92 ± 0.37) and (8.33 ± 0.82) h, MRT (5.31 ± 0.35) and (16.13 ± 1.18) h, respectively. The relative bioavailability of HA-GB-SLN was (157.67 ± 4.85)%. The main pharmacokinetic parameters of ginkgolide B in HA-GB-SLN were as follows: t1/2 (2.81 ± 0.35) h, ρmax (45.91 ± 2.89) ng · mL-1, tmax (1.58 ± 0.02) h, MRT (6.96 ± 0.33) h, AUC0-18(510.79 ± 17.77) ng · h · mL-1, AUC0-∞ (525.45 ± 16.68) ng · h · mL-1. CONCLUSION: The method is sensitive, accurate, specific, and convenient for the pharmacokinetic study of huperzine A. Compared with huperzine A commercial tablets, solid lipid nanoparticles loaded with huperzine A and ginkgolide B have better absorption, shorter onset time, and significantly improved bioavailability.

Identification of a metal ion-induced degradation impurity in huperzine A injection / 中国药学杂志

OBJECTIVE: To identify the structure of an impurity of huperzine A injection. METHODS: HPLC-QTOF-MS was adopted to analyze the structure of the impurity, which was further isolated and prepared by prep-HPLC and the structure was established on the basis of spectroscopic data. RESULTS: The impurity was elucidated as (111E)-7,8-dichloro-5-amino-11-ethylidene-7-methyl-5,8,9,10-tetrahydro-5,9-methenecyclooctatriene [b] pyridin-2(1H)-one. CONCLUSION: The impurity was deduced as a metal ion-induced degradation product. This investigation provides reference for the improvement of quality control and process optimization of huperzine A injection.

Identification of alkaloids and huperzine A-producing endophytic fungi isolated from wild Huperzia serrata / 国际药学研究杂志

Objective To screen endophytic fungal strains isolated from wild Huperzia serrata which can produce alkaloids and huperzine A (HupA). Methods Thirty-three endophytic fungal strains were obtained from H.serrata.Alkaloid production was assayed with alkaloid precipitation and acid dye colorimetry. Then, the crude extracts of alkaloids produced from fungal strains were analyzed by modulation of acetylcholinesterase(AChE) activity, TLC and HPLC. Identification of endophytic fungi producing HupA was based on morphology analysis. Results Seven alkaloids produced by endophytic fungal strains were determined based on alkaloid precipitation and acid dye colorimetry, among which strain V generated HupA in vitro. Morphology analysis showed that strain V belonged to Penicillium. Conclusion A high percentage of fungal strains isolated from H.serrata leaves has alkaloid producing potentials. It is the first report that Penicillium species produced HupA. The screening of endophytic fungi producing HupA based on alkaloid precipitation and acid dye colorimetry, AChE activity and structure identification are more efficient and directive.

Transcellular transport characteristics of huperzine alone or in combination with ginkgolide B across Caco-2 and Madin-Darby canine kidney cell monolayer

Objective: To study the various processes involved in transcellular transport (TT) of huperzine A alone or in combination with ginkgolide B in Caco-2 and Madin-Darby canine renal (MDCK) cell monolayer. Methods: The transepithelial passage was assayed in the apical-to-basolateral (AP to BL) direction and opposite direction (BL to AP) in both cell lines. The determination of huperzine A and ginkgolide B were performed by high performance liquid chromatography (HPLC). The passage rates of huperzine A and ginkgolide B were calculated. Bi-directional TT (absorption and secretion) were taken in huperzine A and ginkgolide B in Caco-2 and MDCK cell monolayer. Results: TT absorption and secretion kinetics of huperzine A and ginkgolide B across two cells existed at the same time. The passage rates of huperzine A were increased significantly with adding different concentrations of ginkgolide B. Conclusions: The compound preparations of HA in combination with GB for dementia caused by cerebral ischemic have synergistic effects on the pharmacodynamics, and improve the bioavailability through BBB.