Chemical Constituents And Pharmacognostical Identification Of Vitex Negundo Var. Cannabifolia Fruits

The dry and mature fruits of Vitex negundo L. var. cannabifolia (Sieb. Et Zucc.) Hand.-Mazz. (VC) are called "mu jing zi" in Chinese, which have effects of dispelling phlegm and eliminating dampness, relieving cough and asthma, and regulating qi-flowing and relieving pain. It’s reported by modern pharmacological studies that VC possess the activities of relieving bronchial smooth muscle, analgesic and anti-inflammatory, antioxidant, and endocrine regulation. In order to better develope and utilize VC, we thus perform systematic chemical investigation on VC fruits in order to provide substantial basis for the bioactive components elucidation. In addition, due to the mix use of VC fruits and Vitex negundo (VN) fruits in folk medicine, our work also intents to confirm the similarities and differences between them based on five traditional identification methods of pharmacognosy (identification of original plant, macroscopical identification, microscopical identification, physical and chemical identification, and identification by DNA molecular marker). Our work is therefore consisted the following two parts:1. Study on the chemical constituents of VC fruitsThe ethanol extract of VC fruits was suspended in H2O and partitioned sequentially with petroleum ether, CH2Cl2, EtOAc and n-butanol, respectively, to give five fractions. Based on modern chromatographic techniques and spectroscopic analysis, We have obtained and identified 6 compounds from the petroleum ether-soluble part and 19 compounds from the CH2Cl2-soluble part. A total of 25 constituents were obtained, among which there were 9 lignans,4 diterpenoids,1 triterpene,2 steroids,6 flavonoids, and 3 phenolic acid compounds. Compounds VCL-8,12,14 were isolated from VC for the first time, identified as: 4β-hydroxyasarinin, Abietatriene-3β-ol and Friedelan, respectively.2. Comparative pharmacognostical identification of VC fruits and VN fruitsVC is a variety of VN. Both of their plant and fruits showed high similarity to each other, which therefore resulted in the mix use. Comparative pharmacognostical identification of the two fruits has been done based on five traditional identification methods of pharmacognosy (identification of original plant, macroscopical identification, microscopical identification, physical and chemical identification, and identification by DNA molecular marker). In the plant origin identification, the major difference was observed for the leaves serration. VN leaves are often entire, whereas VC leaves have incised margins. In the morphological identification, no obvious difference were found, while in the microscopic identification of the fruits powder, the major difference was the characteristics of the sclereids. Sclereids of VC fruits are usually oval and ellipsoid with thin xylans and bigger and ellipsoid lumens, while the sclereids of VN fruits are usually needle and wedge-shaped, with thick xylans and long and narrow lumens. In the physical and chemical identification, LC-MS analysis, combined with the established chemical constituents of the genus Vitex, was employed to reveal that a total of 115 and 79 deduced compounds were found in VN and VC fruits, respectively, among which 69 are shared by these two medicinal fruits, suggesting their high chemical similarities. In the molecular identification, the fresh leaves of VC and VN were used to extract DNA and ITS2 sequences were amplified through ITS2 general primers. Based on the obtained ITS2 sequences, the identification between VC and VN were performed. The results showed that both of the ITS2 sequences are of 221 base pairs (bp), but with 6 different bp (No.26,36,160,163,171 and 173), indicating that ITS2 sequences can be used as an effective mean of identification between VC and VN.In conclusion, a total of 25 chemical compounds were isolated and identified from VC fruits, with lignans as the major constituents and 3 compounds first reported for VC, which enriched the chemical data of VC. Through the comparative pharmacognostical identification of VC fruits and VN fruits, we suggest that microscopic identification of powder and ITS2 sequences identification are effective methods for identification and can also provide a basis for the identification of Vitex species. LC-MS analysis revealed the high similaritied between VC and VN fruits, thus providing some evidence for the mix use of these two medicinal fruits. However, scientific record of these two fruits as one medicinal resource still needs further systematic chemical and pharmacological studies.