| Santalum album L. is belonged to family Santalaceae and genus Santalum. The heart wood of Santalum album which is rich in essential oil is widely used in Perfume Industry, carving Industry and medical and health care. Santalum album is ererygreen, having dense branches and leaves which have a fast regeneration. But there are still no reports about chemical constituents and rational utilization of leaves of Santalum album. The present study is to make some basic research for the Utilization of the leaves.1. Study of flavonoids from the leaves of Santalum albumThe leaves of santanlum album were extracted with 70% ethanol. The extration was then purified with macroporous adsorptive resins(AB-8), polyamide and Sephadex LH-20. Eight compounds were obtained as viceninⅡ, orientin, isoorientin, vitexin, isovitexin, chrysin-8-C-β-D-glucopyranoside, chrysin-6-C-β-D-glucopyranoside, isorhamnetin. All of these compounds were first isolated and identified from the speices.2. HPLC fingerprint study of the leaves of santalum album2.1 The opitimal chromatography conditionAfter systematic experiments, the opitimal chromatography condition was established. The separation was achieved on a C18 column(5μm,250 mmx4.6 mm. Agilent, USA) with a mobile phase consisting of methanol-0.5% acetic acid. The procedure for gradient elution was 22%B for 0-18min,22%B-30%B for18-20 min,30%B-31.5% B for 20-35 min,31.5% B for 35-42 min,31.5% B-40% B for 42-44 min,40% B for 44-56 min,40% B-60% B for 56-65 min. The Flow rate was 1 ml/min. the column temperature was maintained at 30℃. The detection wavelength was at 338 nm2.2 The methodological studyThe stability, repeatability and precision of the HPLC method were studied and the result showed the RSD of relative retention time (RRT) and the relative peak area (RA) were less than 10%, which was meet the requirements of HPLC fingerprint.2.3 The analysis and evaluation on HPLC fingerprint of Santalum album leavesThe HPLC fingerprint of 10 batch of Santalum album L. were investigated, which was referred to the technical demand of the study about injection of traditional Chinese medicine and the technical demand of fingerprint about injection of traditional Chinese medicine established by the State Food and Drug Administration.13 characteristic common peak peaks were established and five peaks were identified by comparing with the reference standards. The confirmed peaks were listed as following:peak 6 for Vicenin-2, peak 8 for orientin, peak 10 for isoorientin, peak 11 for vitexin, peak 12 for isovitexin. The Peak 12 was assigned as the reference and all common peaks were compared with it to establish the RRT and RA. The results of 10 batch samples showed the RSD% of RRT of the common peaks were less than 3%, the RSD of RA were larger, and the area of non-common peaks accounted for total peak area was less than 10%. The research of HPLC fingerprint for leaves of santalum album will provide a new method for its quality control.3 Study on Quality Standards of Santalum album leaves3.1 Shape and propertiesThe leaves of Santalum album were collected from different origin at different time period. The characters of the leaf is summarized as following:abottle green, ellipse ovoid, membranous or thinly leathery, smooth,4-5 cm long and 2-4 cm wide, apex mucronate or acute mucronate, base cuneate, net vein and raised midrib on the undersides of the leaves, slender petiole and 1-1.5cm long, tiny odor and little bitter in taste.3.2 Identification3.2.1 Microscopical chatacteristicsThe identification items of the leaves include tissue signature, surface characteristics and powder characteristicsCharacters of leaf transaction:the epidermal cell is square and exine is incrassate and covered with thinner stratum corneum. The vascular is collateral bundle with one to several layers of collenchymas surrounded. The vascular cambium is clear. There is no differentiation of palisade and spongy parenchyma and the mesophyll tissues are composed with round or oval-shaped thin-walled cells, tightly packed. There are large amounts of cluster crystal surrounded the vein and decreased gradually with the vein extending.3.2.2 Surface characteristics;Surface characteristics:cells of leaf upper epidermises are square or polygonal, closely packed and no stomata distribution, no trichome. The cells of lower epidermis are polygon with a large number of stomas distributed. The stomata are paracytic type. The number of stomata per unit area (mm2) is 622.2, stomatal index is 0.28, and vein-islet number is 27.8-33.3. A large number of cluster crystals are surrounded the main and lateral veins, which decreased with the veins weakened.3.2.3 Powder CharacteristicsThe powder is dark green and has more clustered crystals with more sharp edges, the diameter of which is 10-15μm. Most of these crystals are distributed in the parenchyma cells adjacent to the vein; epidermal cells polygonal, tightly packed. Stomas are paracytic type.3.2.4 TLC IdentificationsThe leaves of santalum album are dried and crushed to powder.0.5 g powder was mixed with 20 ml alcohol, and then extracted with ultrasonic assistance for 15 min. the test sample was obtained by filtration of the extraction. Reference solution was prepared in concentration of 0.1 mg/ml by dissolving reference substance of vitexin and isovitexin.5 ul sample solution and 2 ul reference solution were drawed respectively with capillary tube and applied to the same polyamide lamella, then developed with methanol-acetic ether-water(3:1:2). The lamellar was sprayed with 5% AlCl3 ethanol solution, then dried in 105℃to that spot can be seen clearly under the ultraviolet lamp of 365 nm. The extraction and the reference solution should have same fluorescent spots.3.3 total ash determinationThe method was according to appendix IX K of Chinese Pharmacopeia (2010 Edith). The total ash was 6.97%.3.4 Content Determination3.4.1. Chromatographic conditionThe HPLC column is TC C18 column (5μm,250 mm×4.6 mm. Agilent, USA). The separation was carried out with the mobile phase consisting of methanol-0.5% acetum (40:60, v/v) at a flow rate of 1.0 ml/min. The effluent was monitored at a wavelength of 338 nm and the temperature of column was kept at 30℃. The sample injection volume was 5μl.3.4.2 Preparation of samplesThe leaves of santalum album were dried at 60℃, then crushed to powder and sieved with 40 mesh sieve.1 g powder was precisely weighed and extracted with 50 ml 70% ethanol with ultrasonic assistance for 40 min. the solution was cooled to room temperature and filtered with 0.45μm filters, which was the test sample.3.4.3 Method validationThe linearity and extent of vitexin and isovitexin was y= 3.9253x+31.797, R2= 0.995,7.625-488μg/ml,y= 4.8079x+29.32, R2= 0.999,8.047-515μg/ml,respectively. The RSD% of precision was 0.62%与0.63% respectively. The solution was tested at 2、4、8、12、24 h for stability study. RSD of the content was 3.1% and 2.8%. The recovery of vitexin and isovitexin at high, middle and low concentration was 95.55%/95.86%, 96.59%/96.95%,96.76%/97.13%, respectively.3.4.4 Sample determinationThree batch samples collected from Zhanjiang south medicine farm were determined with the validated methods. The result showed that the content of vitexin and isovitexin was 1.13-1.25 mg/g and 1.2-1.46 mg/g, respectively.4. Study on the dynamic variation law of the contents of seven flavonoid glycoside from santalum album4.1 The development of HPLC method for simultaneous determination of seven flavonoid glycosideThe linearity and extent of seven flavonoid glycoside was y= 919.05x-25.556, R2=0.9993,0.02098-2.1698mg (viceninⅡ); y= 1205x-22.362, R2=0.9994, 0.01987-1.9874(orientin);y=1334.4x-31.797, R2=0.995,0.02058-2.0580(isoorientin); y= 1728.2x-17.211, R2=0.995,0.02041-2.0408 (vitexin); y= 2231.9x-40.665, R2=0.995,0.02152-2.1521 (isovitexin); y= 1108.3x-6.1395, R2=0.996,0.01084-1.0849 (chrysin-8-C-β-D-glucopyranoside); y=2883.4x-25.446, R2=0.9970.009842-0.9842 (chrysin-6-C-β-D-glucopyranoside),respectively. The RSD of precision was 0.62%与0.63% respectively. The solution was test at 2,4,8,12,24 h for stability study. RSD of the content was 3.1% and 2.8%. the recovery of vitexin and isovitexin at high, middle and low concentration was 95.55%/95.86%,96.59%/96.95%,96.76%/97.13%, respectively. The RSD of precision of each determinand is below 4%, RSD of repeatability is below 5%. Stability experiments showed all flavonoid glycoside are stable in 24 h(RSD<5%). The recovery of all flavonoid glycoside is in 95%-97%.The contents of the seven compounds in the leaves from high to low were isovitexin, vitexin, orientin, isoorientin, viceninⅡ, chrysin-8-C-β-D-glucopyranoside, chrysin-6-C-β-D-glucopyranoside. The area of vitexin and isovitexin were accounted for 35-45% and 20-30% of total area (338 nm)4.2 Content determination of leaves of santalum album in different collection periodContent determination were carried out on samples collected from 12 month, the results showed the total flavonoids were accounted for 3-7% and the lowest content was about 3.3% in 2-3 month, highest content was about 6.2% in 8-10 month. The content was about 5% in December, which was almost same with that in January of next year. The content was highest in 8-10 month which was also the rapid growth period. So this period was best for collection. The contents of flavones of five trees are quite different.Content of isomeric compounds was regular changed. The change regularity of viceninⅡwas similar to vitexin-isovitexin. Isomers of Orientin-isoorientin had similar change regularity with vitexin-isovitexin in 1-9 month, but the content of Orientin-isoorientin rised after October and the highest content reached during October to January of next year, which was different to vitexin-isovitexin. We conclude from the result that the key regulatory synthetase for these similar structures has homology and the production of which were in a dynamic balancing state4.3 Variation of flavonoids Content of different leaf ageNoticeable variation of flavonoids Content was showed during April to November. The most notable difference of flavonoids content was during May to October, in which flavonoids in young leaves was two times than that in old leaves. The minor differences of flavonoids content was showed during December to March of next year. We conclude from the results that the flavonoids produced rapidly during leaf early growth stage, then the synthetic speed of flavonoids was lower than the accumulation speed of primary metabolites. 4.4 Variation of flavonoids Content of different tree ageflavonoids content was compared during three age segments:1-6 y,6-10 y, above 10 y. minor difference was show in the first two age segments, but for the third age segment, the flavonoids Content increased markedly, which was two times than that of 1-6 y and 6-10 y. flavonoids Content of trees in "Jiexiang" group was higher than that in "Non-Jiexiang" group4.5 Influence of ethephon induction to treesThe flavonoids content of trees before induction and after induction for one month was lower than that of trees in blank control group. The flavonoids content increased 2-4 times when ethephon was applied for eight weeks and 3% ethephon induction group>2% ethephon induction group> solvent blank control group. We inferred that it may be the stress reaction of the plant to the stimulation of exogenous substances, which caused the increase of production or activities of flavonoids synthase. It also may be the ethephon which inhibiting their growth of the leaves, so the content of flavonoids was relatively higher.4.6 influence of drying temperature and decay to the flavonoids content of leavesFlavonoids content of leaves was compared before and after dry in 60℃and fresh and decayed leaves. The results showed no obvious changes could be found between fresh and dry leaves, but decay made the Flavonoids content decreased about 50%. So we conclude that leaves can be dried in 60℃but corrosion prevenation was needed.5. Studies on the extraction and separation technology of vitexin and isovitexin from the leaves of santalum albumThe aim of the research was to establish an optimized technology for extraction and separation of vitexin and isovitexin from santalum album5.1 technology of extractionThe influence factors of extraction rate of vitexin and isovitexin were investigated including the extraction methods, ethanol concentration of the extract, extraction temperature and extraction times. the results showed that the best extracting method and conditions were determined to be ultrasonic extraction with 70% ethanol at 50℃for two times and each extracting time was 40 minutes.5.2 Technology for purification.The technology for purification of vitexin and isovitexin was investigated including dynamic adsorption rate of different macroporous adsorption resins, desorption rate and purity of crude extracts with different eluent. The optimized method for purification of vitexin and isovitexin were listed as following:AB-8 macroporous absorption resin, sample concentration was 0.5 g/ml(dry herbs), the volume of sample loading was less than 6.6%(crude drug/resin) and 1.49%(Total quantity of vitexin and isovitexin/resin), the order of eluting was 10% ethanol was for 10-12BV(until the effluent colourless) then to change to 30% ethanol for 4-5BV(the desorption rate>85%). The purity of vitexin and isovitexin was 5.85% and 20.41% respectively.6. HPLC study of pharmacokinetics and tissue distribution of isovitexin in ratsA HPLC method for isovitexin determination in rat plasma and different tissues was developed. The separation was achieved on a C18 column with a mobile phase consisting of methanol-1% acetic acid (40:60, v/v) at a detection wavelength of 338 nm and a column temperature of 30℃. Rutin was chosen as internal standard. The blood samples for pharmacokinetic study or the tissue samples for distribution study was collected at fixed time interval after the rats received isovitexin intravenously. The linear range of the standard curves was 0.19-99μg/mL in plasma and 0.024-3.09μg/mL in tissues. The LOQ was 0.19μg/ml in plasma and 0.024μg/ml in tissues. Relative recoveries of isovitexin ranged from 93% to 105% in plasma and 87% to 112% in tissues. The intra-and inter-day precisions were all below 8%. The pharmacokinetic process of isovitexin in three different dosage was fitted a two-compartment open model in rat. Tissue distribution study show that the sequence of tissue drug concentration from high to low was kidney> liver> lung≈ovary> heart≈spleen> brain. |
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