Unique features of the m6A methylome and its response to drought stress in sea buckthorn (Hippophae rhamnoides Linn.).

In plants, recent studies have revealed that N6-methyladenosine (m6A) methylation of mRNA has potential regulatory functions of this mRNA modification in many biological processes. m6A methyltransferase, m6A demethylase and m6A-binding proteins can cause differential phenotypes, indicating that m6A may have critical roles in the plant. In this study, we depicted the m6A map of sea buckthorn (Hippophae rhamnoides Linn.) transcriptome. Similar to A. thaliana, m6A sites of sea buckthorn transcriptome is significantly enriched around the stop codon and within 3'-untranslated regions (3'UTR). Gene ontology analysis shows that the m6A modification genes are associated with metabolic biosynthesis. In addition, we identified 13,287 different m6A peaks (DMPs) between leaf under drought (TR) and control (CK) treatment. It reveals that m6A has a high level of conservation and has a positive correlation with mRNA abundance in plants. GO and KEGG enrichment results showed that DMP modification DEGs in TR were particularly associated with ABA biosynthesis. Interestingly, our results showed three m6A demethylase (HrALKBH10B, HrALKBH10C and HrALKBH10D) genes were significantly increased following drought stress, which indicated that it may contributed the decreased m6A levels. This exhaustive m6A map provides a basis and resource for the further functional study of mRNA m6A modification in abiotic stress.

DNA barcoding based identification of Hippophae species and authentication of commercial products by high resolution melting analysis.

Sea buckthorn (Hippophae), an ancient crop with modern virtues, is increasingly consumed in source of foods and nutraceuticals. The growing demand leads to the adulteration of commercial sea buckthorn products, which is a common form of food fraud. Herein, a high resolution melting assay, targeting a DNA barcoding region of the internal transcribed spacer 2 (ITS2) (Bar-HRM) was developed to identify the seven native Chinese Hippophae species, and to authenticate commercial sea buckthorn products. Melting data from the HRM assay demonstrated that all Hippophae species could be clearly distinguished. Then, application to commercial sea buckthorn products indicated the existence of adulterants or contamination, further confirmed using Sanger sequencing results for PCR products from HRM. The Bar-HRM technique proposed in this work could provide a method for regulatory agencies, promoting consumers trust, and raise the quality and safety of sea buckthorn products.

Proanthocyanidins in Sea Buckthorn (Hippophaë rhamnoides L.) Berries of Different Origins with Special Reference to the Influence of Genetic Background and Growth Location.

Wild sea buckthorn berries from Finland (Hippophaë rhamnoides ssp. rhamnoides) and China (ssp. sinensis) as well as berries of two varieties of ssp. rhamnoides cultivated in Finland and five of ssp. mongolica cultivated in Canada were compared on the basis of the content and composition of proanthocyanidins (PAs). Among all of the samples, only B-type PAs were found. The contents of dimeric, trimeric, tetrameric, and total PAs were in the range of 1.4-8.9, 1.3-9.5, 1.0-7.1, and 390-1940 mg/100 g of dry weight, respectively. The three subspecies were separated by three validated factors (R(2), 0.724; Q(2), 0.677) in the partial least squares discriminant analysis model. Significant differences in total PAs were found between the ssp. rhamnoides and mongolica samples (p < 0.05). In ssp. rhamnoides, samples grown in northern Finland were characterized by a high amount of total PAs, typically 2-3 times higher than that in the level found in southern Finland. In ssp. sinensis, altitude did not have a systematic effect on the PA composition, suggesting the significance of the interaction between genetic background and growth location.

[Genetic and chemical discrimination of traditional Tibetan medicine seabuckthorn based on DNA barcode and ¹H-NMR metabolic method].

To differentiate three medicinal Hippopahe species of seabuckthorn, a combined genetic and chemical identification method was established in this study. ITS2 and psbA-trnH were tested for identification of 3 species of seabuckthorn. Detection of the kimura 2-parameter (K2P) distance, the neighbor-joining (NJ) tree and the barcoding gap were used to assess the identification efficiency. ¹H-NMR based metabolic method was applied to acquire the profile of metabolites. PCA was used to analysis the metabolite data. The results indicated that DNA barcode combined ¹H-NMR based metabolic method is a powerful tool for the identification of 3 medicinal Hippopahe species of seabuckthorn. The finding demonstrated that different genetic variation and chemical constituents existed among 3 medicinal Hippopahe species of seabuckthorn. The combined identification method will improve the reliability of species discrimination and could be applicable to much other ethnic medicine which has various origins in China.

A new strategy for complete identification of sea buckthorn cultivars by using random amplified polymorphic DNA markers.

DNA fingerprinting is both a popular and important technique with several advantages in plant cultivar identification. However, this technique has not been used widely and efficiently in practical plant identification because the analysis and recording of data generated from fingerprinting and genotyping are tedious and difficult. We developed a novel approach known as a cultivar identification diagram (CID) strategy that uses DNA markers to separate plant individuals in a more efficient, practical, and referable manner. A CID was manually constructed and a polymorphic marker was generated from each polymerase chain reaction for sample separation. In this study, 67 important sea buckthorn cultivars cultivated in China were successfully separated with random amplified polymorphic DNA markers using the CID analysis strategy, with only seven 11-nucleotide primers employed. The utilization of the CID of these 67 sea buckthorn cultivars was verified by identifying 2 randomly chosen groups of cultivars among the 67 cultivars. The main advantages of this identification strategy include fewer primers used and separation of all cultivars using the corresponding primers. This sea buckthorn CID was able to separate any sea buckthorn cultivars among the 67 studied, which is useful for sea buckthorn cultivar identification, cultivar-right-protection, and for the sea buckthorn nursery industry in China.

Carotenoid composition of berries and leaves from six Romanian sea buckthorn (Hippophae rhamnoides L.) varieties.

Berries and leaves from six varieties of Carpathians' sea buckthorn (Hippophae rhamnoides L., ssp. Carpatica) were analysed for their carotenoid composition (free and esterified) using a combination of HPLC-PAD, GC-MS and UHPLC-PAD-ESI-MS techniques. GC-MS techniques revealed the fatty acid profile specific for each berry variety, while targeted UHPLC-MS analysis identified the fatty acids involved in carotenoids esterification: palmitic (C16:0), myristic (C14:0) and stearic (C18:0). Total carotenoid content varied between 53 and 97 mg/100g dry weight in berries, and between 3.5 and 4.2mg/100g DW in leaves. The carotenoid di-esters represented the main fraction among berry varieties having zeaxanthin di-palmitate as major compound, while leaves contained only free carotenoids like lutein, ß-carotene, violaxanthin and neoxanthin. Principal component analysis identified the suitable carotenoid biomarkers characteristic for the Carpathians' sea buckthorn from Romania with contribution to their taxonomic classification and authenticity recognition.

[Phylogenetic relationships of seabuckthorn based on ITS sequences].

Sequences of the internal transcribed spacer (ITS) was compared in three seabuckthrons (Hippophae rhamnoides subsp. sinensis, H. tibetana and H. neurocarpa) distributed in Qinghai Province, then the systematic positions of 15 seabuckthron samples were analyzed with Elaeagnaceae angustifolia being outgroup. The results showed that ITS sequences in three seabuckthron species varied in length from 600 to 605 bp. ITS-1, 5.8S and ITS-2 varied from 201 to 203 bp, 166 to 167 bp and 232 to 236 bp, respectively. The sequence divergence among three seabuckthorn species was also remarkably high; Cluster analysis based on ITS indicated that H. gonicocarpa subsp. litangensis and H. gonicocarpa subsp. goniocarpa were distinct and could be classified as H. gonicocarpa and H. litangensis. H. gyantsensis had the closest genetic relationship with H. salicifolia and the distant relationship with H. rhamnoides subsp. sinensis. The positions of nine subspecies of H. rhamnoides based on morphological classification were different from the results of ITS.

UHPLC/PDA-ESI/MS analysis of the main berry and leaf flavonol glycosides from different Carpathian Hippophaë rhamnoides L. varieties.

INTRODUCTION: Sea buckthorn (Hippophaë rhamnoides L.) is known to be rich in many bioactive compounds (such as vitamins, phenolics, carotenoids) important for human health and nutrition. Among the phenolics, berries and leaves contain a wide range of flavonols that are good quality and authenticity biomarkers. OBJECTIVE: To compare the composition of the main flavonols of Romanian sea buckthorn berry and leaf varieties and to identify the specific biomarkers that contribute to sample differentiation among varieties. MATERIAL AND METHODS: Six varieties of cultivated sea buckthorn (ssp. Carpatica) berries and leaves were analysed by UHPLC/PDA-ESI/MS. RESULTS: Berries and leaves contained mainly isorhamnetin (I) glycosides in different ratios. Whereas I-3-neohesperidoside, I-3-glucoside, I-3-rhamnosylglucoside, I-3-sophoroside-7-rhamnoside and free isorhamnetin were predominant for berries (out of 17 compounds identified), I-3-rhamnosylglucoside, I-3-neohesperidoside, I-3-glucoside, quercetin-3-pentoside, kaempferol-3-rutinoside, and quercetin-3-glucoside were predominant in leaves (out of 19 compounds identified). Berries contained, on average, 917 mg/100 g DW flavonol glycosides. Leaves had higher content of flavonol glycosides than berries, on average 1118 mg/100 g DW. The variation of the quantitative dataset analysed using principal component analysis accounted for 91% of the total variance in the case of berries and 73% in case of leaves, demonstrating a good discrimination among samples. CONCLUSION: Based on quantitative analysis, by principal component analysis, the flavonol derivatives can be considered as biomarkers to discriminate among varieties and to recognise specifically the berry versus leaf composition.

Development, characterization and antioxidant activity of polysorbate based O/W emulsion containing polyphenols derived from Hippophae rhamnoides and Cassia fistula

The objective of this study was to develop a pharmaceutical O/W emulsion containing plant-derived polyphenol extracts and evaluate its stability and antioxidant activity. O/W emulsions were prepared using ionic surfactant polysorbate 80 (Tween 80®). The odorwas adjusted with few drops of blue sea fragrance. DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was used to evaluate the antioxidant activity of the plant extracts alone and emulsions containing these extracts. Physical stability was assessed by submitting the emulsions to storage at 8 ºC, 25 ºC, 40 ºC and 40 ºC + 70% RH (relative humidity) for two months. Various physical characteristics of emulsions monitored, include color, creaming, liquefaction, centrifugation and pH. Brookfield rotational rheometer was used to determined viscosities and rheological behavior of emulsions. Different types of emulsion were determined microscopically, while pH values of emulsions were measured by a pH meter. Electrical conductivity data confirmed that the outer phase was water. Samples presented an acceptable pH value for an external topical use. Shear thinning behaviour was observed for all emulsions. The polyphenol-rich-plant-derived extracts alone and the extract containing emulsions showed good antioxidant activities. This research confirmed that the method used was suitable for preparing emulsions with Hippophae rhamnoids and Cassia fistula extracts, suggesting that those emulsions are suitable for topical use.

O presente estudo objetivou o desenvolvimento de uma emulsão farmacêutica óleo-água contendo extratos de plantas ricos em polifenóis, a comparação à sua formulação-controle e a avaliação de sua estabilidade, assim como de sua capacidade antioxidante. Extrato concentrado de Hippophae rhamnoids e Cassia fistula foi encapsulado no interior da fase oleosa da emulsão O/W. As emulsões foram preparadas usando o tensoativo iônico monooleato sorbital de polioxietileno (Tween 80®). O odor foi ajustado pela adição de algumas gotas de fragrância azul do mar. O ensaio do DPPH (1,1-difenil-2-picrilidrazil) foi utilizado para avaliar a atividade antioxidante dos extratos de plantas sozinhos e nas emulsões contendo os extratos. A estabilidade física foi avaliada submetendo os cremes a diferentes temperaturas de estocagem, como a 8 ºC, 25 ºC e 40 ºC e a 40% + 70% de umidade relativa por um período de 2 meses. As características físicas das emulsões foram monitoradas por 2 meses incluindo cor, cremosidade, liquefação, centrifugação e pH. O reômetro rotacional de Brookfield foi utilizado para determinar a viscosidade e o comportamento reológico das emulsões. O programa Rheocalc Brookfield foi utilizado para análise dos dados. As características organolépticas também foram avaliadas. O tipo de emulsão foi determinado microscopicamente, enquanto o pH das emulsões foi avaliado por meio de um pHmetro. A estabilidade farmacêutica esperada das emulsões foi alcançada dentro dos dois meses de estudo. Os resultados da condutividade elétrica confirmaram que a fase externa da emulsão era composta de água. O pH das amostras estava dentro da normalidade para uso tópico. A emulsão apresentou boa fragrância e pode ser retirada da pele com água após a aplicação, características desejáveis em emulsões O/W. Os extratos vegetais ricos em polifenóis isolados ou nas emulsões apresentaram boa atividade antioxidante. Nossos estudos confirmaram que o método utilizado foi adequado para preparar a emulsão semi-sólida contendo extratos de Hippophae rhamnoids e Cassia fistula. Nossos achados sugerem que emulsões contendo extratos de Hippophae rhamnoids e Cassia fistula são adequados para o uso tópico.

Associations of SRAP markers with dried-shrink disease resistance in a germplasm collection of sea buckthorn (Hippophae L.).

Sea buckthorn (Hippophae L.) is a woody, outcrossing dioecious pioneer plant, being widely planted as a new berry crop with rich nutritional and medicinal compounds. This long-juvenile and long-lived woody plant can be more difficult to cultivate than other crop plants. Dried-shrink disease (DSD) is a dangerous pathogen that destroys sea buckthorn and halts commercial production. We estimated variability of sequence-related amplified polymorphism (SRAP) markers in 77 accessions of 22 sea buckthorn cultivars to seek markers associated with DSD resistance and help to identify potential breeding cultivars. Seventeen SRAP primer combinations generated 289 bands, with a mean of 17 bands per primer combination. At a Dice coefficient of 0.852, the dendrogram generated with 191 polymorphic bands clustered 73 accessions of Hippophae rhamnoides into 2 groups and 4 accessions of Hippophae salicifolia into 1 group. Eleven SRAP markers (Me1-Em3(600), Me1-Em1(680), Me2-Em1(650), Me2-Em1(950), Me3-Em6(1300), Me2-Em6(320), Me2-Em6(400), Me1-Em2(600), Me1-Em1(1200), Me1-Em1(1700), Me2-Em2(250)) were significantly correlated with DSD resistance (P < 0.001). These markers provide a viable option for breeding programs that select lineages with DSD resistance, especially when no other genetic information, such as linkage maps and quantitative trait loci, are available.

Endorsing the declining indigenous ethnobotanical knowledge system of seabuckthorn in Central Himalaya, India.

AIM OF THE STUDY: Based on research findings this study is aimed to generate database on ethnobotanical aspects, sustainable utilization by value addition and awareness generation through outreach programme related to Hippophae salicifolia D. Don. (Elaeagnaceae) in the higher Himalayan zone of Uttarakhand in Central Himalaya, India. MATERIALS AND METHODS: An in-depth survey from June 2004 to July 2006 followed by 480 interviews with the help of semi-structured questionnaires was carried out in 24 Hippophae growing locations in 12 different valleys of Uttarakhand. RESULTS: Plant has immense multipurpose properties and is traditionally utilized for food (20(Min)-90%(Max)), medicine (10(Min)-60%(Max)), veterinary (20(Min)-100%(Max)), fuel (10(Min)-80%(Max)), fencing (20(Min)-80%(Max)), agricultural tools (20(Min)-50%(Max)) and dye mordant (60%). Besides, awareness programmes and value added product demonstration resulted in economical upliftment of local inhabitants of Central Himalaya. CONCLUSION: The present manuscript will certainly provide an ethnobotanical statistics' impact on the modern scientific societies regarding conservation, cultivation and popularization of this underutilized wild edible species at mass scale. Simultaneously, these findings have important connotations in light of upcoming organic food and nutraceutical industries in the country.

[Phytochemical analysis of Hippophae rhamnoides L. fruits from Russian varieties cultivated in Finland and food products containing them].

Results of phytochemical analysis of the berries from six Russian Hippophae rhamnnoides L. varieties (Augustinka, Botanitseskaja, Ljubitelskaja, Vorobjvskaja, Pertsik, Trofimovskaja), cultivated in Finland, are presented. The contents of ascorbic acid (vitamin C), tocopherols (vitamin E), total flavonoid (vitamin P), total carotenoid (vitamin A) and total organic acids were analyzed in unfreezed berries. The same parameters were defined in food products (jelly, sweet sauce and compote) containing fruits of three Hippophae rhamnoides L. varieties (Botanitseskaja, Ljubitelskaja and Trofimovskaja). According to the results, Russian Hippophae rhamnoides L. varieties can be cultivated in South-Finland and different kinds of food products with high quality and concentration of biologically active compounds may be prepared from these berries.

Vitamin C content in sea buckthorn berries (Hippophaë rhamnoides L. ssp. rhamnoides) and related products: a kinetic study on storage stability and the determination of processing effects.

The primary vitamin in sea buckthorn berries is vitamin C containing values of approximately 400 mg/100 g. Processing effects were investigated during juice and concentrate production from sea buckthorn berries (Hippophaë rhamnoides) and storage stability of juices was determined for up to 7 d using berries and juices from 2 different growing areas. During industrial juice production the technological processing of the berries caused a loss of about 5% to 11% total ascorbic acid (TAA) in the generated juice. The production of the concentrated juice resulted in 50% depletion of TAA. Sea buckthorn berries and juice were stored at 6, 25, and 40 degrees C for up to 7 d to investigate the temperature effects on TAA during storage. Analysis of kinetic data suggested that the degradation follows a 1st-order model. The results of the experiments showed that storage of sea buckthorn juices for 7 d at cold temperature (6 degrees C) already resulted in a degradation of TAA of about 11% to 12%.

Influence of ambient and enhanced ultraviolet-B radiation on the plant growth and physiological properties in two contrasting populations of Hippophae rhamnoides.

Two contrasting sea buckthorn (Hippophae rhamnoides L.) populations from low and high altitude regions were employed to investigate the effects of prevailing and enhanced ultraviolet-B (UV-B) radiation on plant growth and physiological properties under a UV-B-enhanced/exclusion system. The experimental design included three UV-B regimes, including excluded (-UVB), near-ambient (NA) and enhanced UV-B (+UVB) radiation. Compared with the control (-UVB), NA caused the formation of smaller but thicker plant leaves in both sea buckthorn populations, paralleled with significant increments of carotenoids and UV-absorbing compounds as well as improved water economy. NA also induced more biomass partition from shoot to root, but CO(2) assimilation rate (A), photosynthetic area and biomass accumulation were unaffected. The low-altitude population seemed sensitive to +UVB, as indicated by the decreases in total biomass, A and ascorbic acid content (Asa, an antioxidant) compared with NA. However, little +UVB effect occurred on the high-altitude population, and we suggest that the higher tolerance of this population could be associated with its specific morphological and physiological characteristics, such as small but thick leaves and high-level of Asa content, as well as its greater physiological modification in response to NA, e.g., increases in protective compounds (carotenoids and UV-absorbing compounds) and improvement in water economy, in comparison to the low-altitude population, which form an effective adaptation strategy to enhanced UV-B stress.

[Genetic diversity of Hippophae rhamnoides L. sub-populations on loess plateau under effects of varying meteorologic conditions].

The genetic diversity and genetic differentiation of eight Hippophae rhamnoides L. populations in eastern Gansu Province, as well as the effects of varying meteorologic conditions on them were assessed by using inter-simple sequence repeat (ISSR) molecular marker method. A total of 240 individuals in the populations were sampled. Using eleven primers, 165 bands were generated, ranging in size from 300 to 1500 bp, and 157 (95.76%) were found to be polymorphic. The analysis of molecular variance (AMOVA) demonstrated that there was a relatively high level (76.5%) of genetic variation within the populations, with the gene differentiation coefficient (Gst) and gene flow being 0.2418 and 1.5675, respectively. Therefore, to protect the gene resources of H. rhamnoides, the individuals within the populations should be first considered. Mantel test showed that genetic distance was significantly positively correlated with geographical distance (r = 0.65, p = 0.002), and regression modeling between genetic diversity and meteorologic factors suggested that there was a significant positive correlation between wind speed during blooming and genetic diversity of H. rhamnoides, illustrating that wind speed in blooming period and geographic distance were the vital factors affecting the genetic diversity of H. rhamnoides population.