Discrimination of Tetrastigma hemsleyanum according to geographical origin by near-infrared spectroscopy combined with a deep learning approach.

Recently, deep learning has presented as a powerful approach to overcome the deficiencies of the conventional biochemical approaches. In this study, a method for discriminating medicinal plant Tetrastigma hemsleyanum from different origins was proposed using near-infrared spectroscopy (NIRS) and deep learning models. Support vector machine (SVM), self-adaptive evolutionary extreme learning machine (SAE-ELM), and convolutional neural network (CNN) were used to process the near-infrared spectral data (4000-5600 cm-1). The results indicated that the average recognition accuracy of SVM on the test set samples (n = 60) reached 90%. The average recognition accuracy of SAE-ELM was 98.3%, while CNN correctly discriminated 100% of T. hemsleyanum from different origins. Notably, CNN avoids tedious redundant data preprocessing and is also able to save the trained model for the next call to achieve rapid detection. As above, this study provides an effective deep learning-based method for discriminating the geographical origins of T. hemsleyanum as well as providing a convenient and satisfactory approach to ensure the famous-region of other medicinal plants.

Molecular authentication of Tetrastigma hemsleyanum from its adulterant species using ISSR, CAPS, and ITS2 barcode.

Tetrastigma hemsleyanum is a rare and endangered herb, which is commercialized as the resource of anti-cancer drugs. Wild T. hemsleyanum plants are on the verge of extinction recently, there are increasing numbers of counterfeits on the market. In the present study, inter-simple sequence repeat (ISSR), Cleaved amplified polymorphic sequence (CAPS), and the internal transcribed spacer region II (ITS2) barcode were used for the first time for the authentication of T. hemsleyanum from its commonly counterfeits. ISSR analysis suggested that it was a useful method for distinguishing T. hemsleyanum from its adulterants of different genus. However, it was insufficient to distinguish T. hemsleyanum from those adulterants of the same genus. ITS2 of T. hemsleyanum and the commonly counterfeits were amplified and sequenced. The Neighbor-Joining tree constructed from the ITS2 sequences showed that T. hemsleyanum was clearly differentiated from all counterfeits samples. A mutation site in the ITS2 region of T. hemsleyanum had been found which could be recognized by the restriction endonuclease NcoI. T. hemsleyanum could be readily distinguished from counterfeits as the PCR products from T. hemsleyanum could be digested sufficiently by NcoI, while the PCR products from counterfeits could not be digested. The results indicated that CAPS and ITS2 barcode methods provided effective and accurate identification of T. hemsleyanum from all its adulterants, while ISSR could only distinguish T. hemsleyanum from its adulterants of different genus. The CAPS method developed in the present study will serve as a reliable tool for safe and effective use of T. hemsleyanum in the clinic application. It will also play an important role for the identification, management and conservation of this endangered species.

Estudo de biocompatibilidade in vivo e in vitro do extrato hidroalcoólico de Cissus sicyoides L. - Vitaceae em ratos / Study of in vivo and in vitro biocompatibility of Cissus sicyoides L. - Vitaceae hydroalcoholic extracts in rats

Biocompatibilidade é a capacidade de um material exercer funções específicas quando aplicado em contato com tecidos vivos de determinado hospedeiro, sem, contudo, causar danos ou prejuízo ao mesmo. Este trabalho objetivou determinar a biocompatibilidade in vivo e in vitro do extrato hidroalcoólico do Cissus sicyoides L - Vitaceae. Foram utilizados 30 ratos (Rattus novergicus albinus wistar), com idade entre 45 e 90 dias e pesando entre 170 e 260 g. Os animais foram divididos em 3 grupos (A1, A2 e A3) de 6 animais cada para o teste in vivo, os quais foram sacrificados com 2, 4 e 6 dias, respectivamente. Para o teste in vitro, foram utilizados 12 animais para obtenção do índice de aderência e da capacidade fagocítica dos macrófagos de ratos do grupo controle e do grupo experimental. Nos resultados encontrados no teste in vivo, conclui-se que o extrato apresentou-se biocompatível, visto que não provocou alterações significativas no tecido. Já no teste in vitro, o mesmo não se apresentou biocompatível, pois o extrato puro apresentou índice de aderência baixo (7,1) e taxa de fagocitose elevada (35,7), indicando diferença significante quando comparado ao controle. Porém, quando diluído, o extrato se mostrou inócuo, devido ao aumento dos valores do índice de aderência nas concentrações de 1/10 (61,4) e 1/100 (74,3) nos ensaios, as quais não apresentaram diferença significante quando comparadas ao controle. Após a análise dos dados, concluiu-se que a solução diluída do extrato hidroalcoólico do Cissus sicyoides L. não causa danos ou prejuízos. Entretanto, como nem todos os efeitos farmacológicos foram testados no presente trabalho, não se pode inferir automaticamente que ele é biocompatível em todos os casos.

Biocompatibility is the ability of a material to perform specifictasks when applied to living tissues without causing damage or injuries to it. Thus, this study aimed at determining the in vivo and in vitro biocompatibility of Cissus sicyoides L. - Vitaceae hydroalcoholic extracts. A total of 30 rats (Rattus norvegicus Albinus Wistar), with ages ranging from 45 to 90 days and weighing between 170 and 260g were used. The animals were divided into 3 groups (A1, A2 and A3) with 6 animals each, for the in vivo test, which were sacrificed after 2, 4 and 6 days, respectively. For in vitro test, 12 animals were used to obtain the index of adherence and phagocytic ability of macrophages of rats from the control and the experimental groups. In results found for the in vivo test, it was concluded that the extract was biocompatible, whereas no significant changes were observed in the tissue. As to the in vitro test, the extract was not biocompatible, since the pure extract showed a low rate of adherence (7.1) and a high rate of phagocytosis (35.7), indicating a significant difference when compared to the control group. However, when diluted, the extract was shown to be harmless, due to an increase in the values of the adherence index at the following concentrations : 1/10 (61.4) and 1/100 (74.3) in the tests, which showed no significant differences when compared to the control group. After analyzing the data, it was concluded that since the infusion of the plant is a kind of dilution, its use does not cause any harm to the body. A new study is necessary at the moment to possibily demonstrate its effects on the long term.

[Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

OBJECTIVE: To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. METHODS: Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. RESULTS: There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. CONCLUSION: RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

[Identification of Gynostemma from their adulterant Cayratia japonica by PCR-RFLP].

OBJECTIVE: To establish a convenient and effective method for the identification of Gynostemma and Cayratia japonica. METHOD: Eight species, including Gynostemm pentaphyllum, G. pentagynum, G. cardiospermum, G. longipe, G. yixingense, G. laxiflorum, G. guangxiense and C. japonica were investigated through PCR - RFLP of six chloroplast DNA fragments. The six gene fragments were digested by six restriction endonuclease respectively, including Taq I, Hpa II, EcoR I, Rsa I, Hha I, Hind III. RESULT: Seven species of Gynostemma and their adulterant could be identified by trnK1f-trnK2r and Rsa. CONCLUSION: PCR - RFLP provides a quick, reliable molecular marker technique for identification of Cynostemma and their adulterant Cayratia japonica.