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ORIGINAL ARTICLE
Year : 2020  |  Volume : 12  |  Issue : 4  |  Page : 450-454  

Identification, preliminary genetic, and biochemical analyses the Hedera plants which naturally distribute in Vietnam


1 Department of Extraction Technology and Department of Medicinal Resources, National Institute of Medicinal Materials, Hanoi, Vietnam
2 Department of Basic Sciences in Medicine and Pharmacy, VNUH-University of Medicine and Pharmacy, Hanoi, Vietnam
3 Department of Biotechnology, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam

Date of Submission07-Jul-2020
Date of Acceptance18-Sep-2020
Date of Web Publication23-Jan-2021

Correspondence Address:
Dr. Dinh Doan Long
144 Xuan Thuy Street, Cau Giay District, Hanoi
Vietnam
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pr.pr_65_20

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   Abstract 


Background: Hedera is a genus of 12–15 species of evergreen climbing woody plants in the family Araliaceae that are considered as traditional medicinal plants. Objectives: The objectives were to classify Hedera species collected from different areas in Vietnam and characterize some chemical compounds of these species. Materials and Methods: In this study, the DNA-based technique was used to classify and analyze the genetic source of 21 Hedera plant samples found in Vietnam. In addition, high-performance liquid chromatography (HPLC) is also employed to assess the chemical compounds of these plants. Results: The collected plants mainly distribute into three groups (named as N1, N2, and N3). Based on the phenotypic identification and genetic analysis, the N1 group is indicated as Hedera nepalensis (H. nepalensis), while N3 is Hedera helix (H. helix). The HPLC analysis indicates that most of them contained more hederacoside C than α-hederin. Moreover, N1 plants are able to produce hederacoside C as N3 even the accumulation levels in N1 are moderately lower than those in N3 plants. Conclusion: These results provide important information about the local Hedera plants and help us to select the good medicinal sources for further applications in biomedical sciences.




Keywords: Hedera plants, hederacoside C, high-performance liquid chromatography detection, ITS gene, matK gene, α-hederin


How to cite this article:
Hiep NT, Nhung PT, Nguyen NH, Linh DT, Duong HT, Hang DT, Huyen PT, Thom VT, Khoi NM, Long DD. Identification, preliminary genetic, and biochemical analyses the Hedera plants which naturally distribute in Vietnam. Phcog Res 2020;12:450-4

How to cite this URL:
Hiep NT, Nhung PT, Nguyen NH, Linh DT, Duong HT, Hang DT, Huyen PT, Thom VT, Khoi NM, Long DD. Identification, preliminary genetic, and biochemical analyses the Hedera plants which naturally distribute in Vietnam. Phcog Res [serial online] 2020 [cited 2021 Mar 9];12:450-4. Available from: http://www.phcogres.com/text.asp?2020/12/4/450/307653




   Summary Top


  • Hedera plants which were distributed naturally in several northern mountainous provinces of Vietnam were identified as H. nepalensis var. sinensis
  • matK and ITS markers were performed
  • Hederacoside C and alpha hederin, the active compounds with promising effects, were found in Hedera nepalensis




Abbreviations Used: ASE: Accelerated solvent extraction, BLAST: Basic local alignment search tool, GBSSI: Granule bound starch synthase, HPLC: High-performance liquid chromatography, ITS: Internal transcribed spacer, matK: Megakaryocyte-associated tyrosine kinase, PCR: Polymerase chain reaction.


   Introduction Top


Hedera is a genus of the Araliaceae family that has 15 registered species. In terms of pharmacology, two species including Hedera helix and Hedera nepalensis are the most widely used and studied. H. helix is mainly found in Europe and parts of South Asia, while H. nepalensis distributes in several countries in Europe, the Himalayas, and some high mountain areas of Vietnam.[1],[2] The extracts and products of H. helix and H. nepalensis have been found to positively function in antifungal, treat respiratory diseases, and support diabetes treatment. Most pharmacological studies involving H. nepalensis showed that this species has similar medicinal effects to H. helix. For example, Uddin et al. found that ethyl acetate extracts of H. nepalensis leaf also have antibacterial effects.[3] Besides, the ethyl acetate fraction of H. nepalensis has been documented to work as an antioxidant corresponding to the high content of flavonoid and phenolic compounds.[1] A previous study revealed that the Hedera genus contains various natural compounds such as triterpene saponins, flavonoids, polyacetylenes, and phenolic compounds.[4] In particular, triterpene saponins (most of which are hederacoside C and α-hederin) have been shown to work in bronchospasm, anti-inflammatory, anti-infection, liver protection, antioxidant, and hypoglycemia.[5] Few studies have investigated the classification and genetic variation of H. helix;[6] although this species has not been found in nature in Vietnam, this species has been imported to some localities such as Da Lat and Lam Dong. Meanwhile, in the last few years, H. nepalensis has been discovered in mountainous areas of northern Vietnam. Some preliminary assessments of the chemical and pharmacological properties suggest that H. nepalensis has a correlation and much potential to replace the imported H. helix. At the DNA level, the variety of Hedera plant species can be identified and evaluated by molecular markers.[6]

In the present work, twenty-one samples of Hedera plants which are naturally distributed in Vietnam were collected and these samples were then classified based on phenotypic characterizations as well as genetic information. The HPLC method was used to quantitatively measure the hederacoside C and α-hederin in these Hedera leaf samples. Taken together, we identified the correlation between the content of the main active ingredients and the genotypes, contributing more information to help for the selection of excellent genetic resources for exploitation and further applications in biomedical sciences.


   Materials and Methods Top


Plant materials

Twenty-one Hedera plant samples were identified and collected by the Vietnam National Institute of Medicinal Materials from different places in Vietnam as indicated in [Table 1]. Based on the phenotypic characterization, these plants were firstly divided to be two groups including H. helix (TL1_180_20 and 21) and H. nepalensis var. sinensis (TL1_180_1-19) [Figure 1]. Fresh leaves from the 21 plants were harvested randomly. Each sample was divided into two parts. One was stored in a deep-freezer (-80°C) and used for DNA extraction, whereas the other was dried in an oven at 60°C and ground for HPLC analysis.
Table 1: List of 21 Hedera plants which were harvested in different locations in Vietnam and used for the current study

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Figure 1: Sample pictures (a) TL1_180_21 (Hedera helix); (b) TL1_180_19 (Hedera nepalensis)

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DNA extraction, polymerase chain reaction, and sequencing

Partial matK was amplified by polymerase chain reactions (PCRs) with the primers matK-390F and matK-1326R.[7] The ITS region was amplified by PCR with ITS-17SE and ITS-28SE.[8] The primers were synthesized by PhuSa Biochem Ltd., (Vietnam). Amplification was of 25 μL containing 1 x HF buffer, 0.2 mM dNTPs mix, 0.02 u/μl Phusion DNA polymerase (Thermo Fisher Scientific, USA), 0.3 μM each of two primers and 10–25 ng DNA template. For 2 genes PCR, cycling conditions were: 98°C for 30 s; followed by 35 cycles of 98°C for 10 s 55.1°C for 30 s and 72°C for 30 s and a final extension at 72°C for 10 min. The amplified products were separated in 1.5' (w/v) agarose gels in 1x Tris-acetate-EDTA buffer. The PCR samples were purified and sequenced at First Base Laboratories (Malaysia).

Phylogenetic analysis

DNA sequences tested, refined through Sequencher versatile 5.4.6 and alignment with BioEdit version 7.2.6.1 software. The trimmed sequence was compared (in NCBI-BLAST) with other available Hedera-ITS and Hedera-matK sequences in the GenBank.

Phylogenetic trees of 21 ITS and matK sequences were constructed using MEGAX software based on maximum parsimony and maximum likelihood methods. The bootstrap method or statistically sampling method was chosen for assigning measures of accuracy of phylogenetic tree. Bootstrap analysis with 1000 replicates was conducted to estimate nodal support.

High-performance liquid chromatography analysis

Reference solution

Methanol was added to a certain quantity of hederacoside C and α-hederin standards (98', Sigma-Aldrich, St Louis, MO) to obtain a range of standard solutions with the concentrations of 25, 50, 100, 200, 500, and 1000 μg/ml.

Test solution preparation

An accelerated solvent extraction (ASE) 350 system from Dionex Corporation (Sunnyvale, CA, USA) was used for the extractions. About 2 g of each powered Hedera leaves was homogeneously mixed with 4 g of diatomaceous earth and placed in a 100 ml stainless extraction cell. A stainless steel frit and a cellulose filter (Dionex) were placed at the bottom of the extraction cell to prevent the penetration of fine powder in the collection bottle. The extraction cell was arranged in the cell tray and the samples were extracted under some specific conditions: pressure (1500 psi), fixed volume (100 ml), nitrogen purge time (100 s), and static time (10 min). After extraction, the samples were centrifuged for 10 min at 4000 rpm. The supernatant was collected and filtered through a 0.45 μm nylon filter membrane before injection into the HPLC system. Three replicates of each sample were prepared and detected.

High-performance liquid chromatography conditions

The Shimadzu SPD-20A system (C18 column; 250 mm × 4.6 mm, 5 μm) (Shimadzu Co., Ltd.) was used for HPLC analysis. A mixture of acetonitrile – 0.02' phosphoric acid solution was used as the mobile phase. The elution mode was a binary, high-pressure gradient system and the elution gradients were: 0–25 min, 20'–60' acetonitrile; 25–30 min, 60'–100' acetonitrile. Other running conditions include the detection wavelength (210 nm), the flow rate (1 ml/min), the injection volume (20 ml), and the column temperature (25°C).


   Results Top


Genetic analysis based on matK gene

The DNA sequences were analyzed using a BLAST search and divided into groups with 100' homogeneous samples. Based on the sequencing results, the plant samples were divided to be three groups (haplotypes) with matK (haplotypes M1 to M3) and seven groups with ITS (haplotypes I1 to I7) [Figure 2]a. To compute the phylogenetic tree from the matK gene, H. helix (AJ319073.1) and H. nepalensis var. sinensis (GQ424360.1) were chosen as reference sequences. Using maximum likelihood methods, matK phylogeny with the highest log likelihood (-1086.95) is shown in [Figure 2]b. The initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. [Figure 2]b shows that 16 samples of haplotype M1 are in the same group as H. nepalensis var. sinensis, while haplotype M3 is H. helix. On the other hand, 3 samples of haplotype M2 seem to be close to group M1.
Figure 2: Group of 100' homogeneous samples (a), matK phylogeny using maximum likelihood methods (b) and ITS phylogeny using maximum parsimony method (c). (b) The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. (c) The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches

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The maximum parsimony tree with ITS gene in [Figure 2]c was obtained using the subtree-pruning-regrafting algorithm. Reference sequences for ITS are H. helix (AM503887.2), H. sinensis GU054623.1), and H. nepalensis var. sinensis (AJ131238.1). ITS phylogeny showed that haplotypes I1, I2, I4, and I5 have high similarities with the H. nepalensis reference sequence, haplotype I3 is more similar to H. sinensis. Meanwhile, I6 and I7 haplotypes are separated into the same group as H. helix. Thus, genetic analysis based on ITS and matK genes showed that they could help distinguish H. helix and H. nepalensis.

Hederacoside C and α-hederin analysis by high-performance liquid chromatography

First, the hederacoside C and α-hederin standards were diluted to six gradients (25, 50, 100, 200, 500, and 1000 μg/ml) and detected by HPLC with described conditions. Next, the linear regression equations were formulated as Y = 2356X + 17365 (r2 = 0.9998) (for hederacoside C) and Y = 5875X + 2620 (r2 = 0.9998) (for α-hederin) (where X = the concentration and Y = the peak area).

The Hedera extracts were also analyzed by the same HPLC system and conditions. Based on the chromatograms, the peak areas of hederacoside C and α-hederin as well as the formulated linear regression equations, the quantities of these two compounds in 21 Hedera extracts were calculated and shown in [Figure 3]. The endogenous contents of both hederacoside C and α-hederin were found to be different among the analyzed Hedera samples and the hederacoside C levels were more abundant than a-hederin.
Figure 3: Hederacoside C and α-hederin contents of 21 Hedera species

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   Discussion Top


Hedera plants have been recognized as a traditional medicine for a long time and are widely used in many countries as potential medical herbal for various diseases. Hashmi et al. found lupeol compound from crude extract of H. nepalensis which showed beneficial effects on antidiabetogenic and anti-Alzheimeric rats via antioxidant defense system.[9] Recently, hederagenin such as sapogenin or saponins received many attentions of scientists due to multiple actions on various diseases pathways such as antitumor, anti-inflammatory, or antiviral infection, as well as neuron and cardiovascular protection.[10] Our study combined both genetic and chemical analysis as core criteria to identify Hedera species. The matK gene has been used for species identification and studies in plant molecular systematic and evolution in various plant species.[11] DNA gene analysis techniques have been developed and applied in studying the genetic diversity of Hedera plants. Ackerfield and Wen detected low levels of nucleotide sequence divergence when they use chloroplast DNA and ITS data.[12] Among Hedera species, H. helix is a well-known plant distributed in Europe as well as Asia, however, H. nepalensis var. sinensis is mostly found in Asia including Vietnam. Out of 21 samples collected, 19 samples in our study were classified into H. nepalensis var. sinensis with matK gene. From the analysis of GBSSI (granule-bound starch synthase) marker, a previous study identified that the ivy plants which naturally grow and distribute in several northern mountainous provinces of Vietnam belong to the H. nepalensis K. Koch species.[13] In line with our study, Wu et al. analyzed 13 species in Araliaceae in China and using whole genome sequencing, it is clearly showed that H. nepalensis var. sinensis closed to Fatsia japonica.[13] However, this study did not identify the active compound in H. nepalensis var. sinensis.

In this study, it is found that Hedera species in Vietnam contain rich of hederacoside C and alpha-hederin compounds which are known to have beneficial effect on anti-inflammatory, especially for acute and chronic respiratory inflammation.[14] Using the HPLC method, extracted from leaves of H. helix grown in Poland and the Czech Republic provided lot of alpha-hederin and hederacoside C in the range from 0.7 to 2.3 mg/100 g extract and 13.9–18.4 mg per 100 g extract, respectively.[15] The concentration of hederacoside C was varied between H. nepalensis haplotypes and reached the highest concentration in haplotype I3 (TL1_180_1 and 2). Besides, the data analysis indicates that H. nepalensis samples are capable of producing hederacoside. Among those groups, groups were identified as H. helix which showed richest contents of hederacoside C with range from 5.7' to 9.4' and alpha-hederin with range from 0.4' to 1.4'. The HPLC results showed that the amount of those active compounds in this study was similar with H. helix grown in Europe. According to the European Pharmacopoeia, dried leaves contain at least 3' of the primary saponin, hederacoside C.[16] Similar H. helix content, hederacoside C in H. nepalensis var. sinensis from sample 1 to sample 12 was at higher level than alpha-hederin obtained from fresh leaf extract. Interestingly, from sample 13 to sample 18, alpha-hederin content was similar or even higher than hederacoside content. Alpha-hederin content in haplotype I5 (TL1_180_14) is more than 1.5 times higher than in the H. helix samples. Furthermore, it is known that hederacoside C is actively metabolized and produces the effect of α-hederin in the body.[17] Recently, the new analysis strategy has been found to discover the metabolic pathway of hederacoside C and the α-hederin biotransformation was observed through deglycosylation reactions by removal of sugar moieties [Figure 4].[18] An interesting paper published by Sieben et al. found that alpha-hederin but not hederacoside C or hederagenin had beneficial effects on cardiovascular disease via beta-2-adrenergic receptors.[19] Recently, alpha-hederin was also proved to have anticancer activities performed in cancer cell line.[18],[20] These findings could be valuable suggestions for using H. nepalensis var. sinensis cultivated from sample 13 to sample 18 in group M1 which is abundant distributed in North Vietnam.
Figure 4: Hederacoside C metabolic pathway through biotransformation

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   Conclusion Top


In conclusion, local Hedera in Vietnam was recognized as H. nepalensis var. sinensis, which primarily distributed in Northern areas in Vietnam. By combining the data from genetic indicators with the analysis of the chemical composition, we can obtain essential information on local Hedera plants. From these results, we could recommend combine matK and ITS genes and hederacoside C analysis in classification of Hedera species in Vietnam. These are fundamental data, contributing to the construction and standardization of medical materials in Vietnam.

Acknowledgements

The study was funded by the Vietnam Ministry of Science and Technology (project code NC-2018/02). The authors sincerely thank Mr. Phan Van Truong (National Institute of Medicinal Materials) for assisting in taking samples for research purposes.

Financial support and sponsorship

The study was funded by the Vietnam Ministry of Science and Technology (Project code NC-2018/02).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Jafri L, Saleem S, Ihsan ul H, Ullah N, Mirza B. In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch. Arab J Chem 2017;10:S3699-706.  Back to cited text no. 1
    
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Ackerfield J, Wen J. Evolution of Hedera (the Ivy Genus, Araliaceae): Insights from Chloroplast DNA Data. Int J Plant Sci 2003;164:593-602.  Back to cited text no. 12
    
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Long DD, Bach NX, Thi Thu Thao N, Thi Hong Nhung P, Thi Le Hang D, Thi Thom V, et al. Comparative analysis of different DNA extraction methods and preliminary analysis of genetic diversity of Hedera nepalensis K. Koch. in Vietnam based on GBSSI Marker. VNU J Sci Med Pharm Sci 2019;35:88-95.  Back to cited text no. 13
    
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Havliková L, Macáková K, Opleta L, Solich P. Rapid determination of α-Hederin and Hederacoside C in extracts of Hedera helix leaves available in the czech republic and poland. Nat Prod Commun 2015;10:1529-31.  Back to cited text no. 15
    
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Wu JJ, Zhou X, Gao J, Peng YQ, Hu SH, Qian YX, et al. The complete chloroplast genome sequence of common Chinese ivy Hedera nepalensis var. sinensis (Araliaceae). Mitochondrial DNA B 2019;4:1881-2.  Back to cited text no. 17
    
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Sieben A, Prenner L, Sorkalla T, Wolf A, Jakobs D, Runkel F, et al. Alpha-hederin, but not hederacoside C and hederagenin from Hedera helix, affects the binding behavior, dynamics, and regulation of beta 2-adrenergic receptors. Biochemistry 2009;48:3477-82.  Back to cited text no. 18
    
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Peeters L, Beirnaert C, Van der Auwera A, Bijttebier S, De Bruyne T, Laukens K, et al. Revelation of the metabolic pathway of hederacoside C using an innovative data analysis strategy for dynamic multiclass biotransformation experiments. J Chromatogr A 2019;1595:240-7.  Back to cited text no. 19
    
20.
Adamska A, Stefanowicz-Hajduk J, Ochocka JR. Alpha-Hederin, the Active Saponin of Nigella sativa, as an Anticancer Agent Inducing Apoptosis in the SKOV-3 Cell Line. Molecules 2019;24:2958.  Back to cited text no. 20
    


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