Morus alba L. (cv Ichinose) (MA) was grown in the hydroponic culture system developed by Morera Co., Ltd. (ex Stream Co., Ltd.) in Nagano, Japan. The growing condition is briefly descried below. The seeds were germinated in a polyurethane foam for 30 days at room temperature 22˚C. The hatched MA (approximately, 1 - 2 cm high) was transplanted to the styrofoam board and set in the cultivating stand equipped with stainless steel pan in that water containing appropriate concentration of commercially available fluid fertilizer (Vegetable Life A, OAT Agrio Co., Ltd., Tokyo Japan) is continuously circulated, and then cultivated at 23˚C under illumination of 12 hr light and dark cycle by white fluorescence lamps as shown in Figure 1.

After 3 months cultivation of MA (HM) by the in-room hydroponics, each of leaf, twig and root were harvested, freeze-dried and powdered using ball-mil. The power was packed in vacuum aluminum bags and kept in a freezer at −20˚C until use.

Practically, the leaves and twigs are harvested repeatedly from the tree maintained until 6 months, and the roots are harvested at 6 month, because the yield of fibrous root markedly reduced after 6 month. For the animal feeding experiments, air dried powders prepared from these samples were used.

The fresh leaves from HM and field grown MA (FM) were dissected into 5 mm in diameter pieces with a biopsy trepan (Kai corporation, Tokyo, Japan), fixed in the Farmer’s fixative (ethanol:glacial acetic acid 3:1), dehydrated in a series of graded ethanol and embedded in paraffin after xylene substitution. The paraffin block is trimmed, sectioned by a microtome to obtain 10 µm thick sections to observe under microscopy after staining by 0.1% toluidine blue.

DNJ was quantified by LCMS as described elsewhere [20]. Briefly, MA sample

powders were extracted with water and the extract was separated on HPLC equipped with CAPCELL CORE PC reverse phase column (Shiseido Co., Ltd, Tokyo) using a gradient elution mode as follows: A 0.1% formic acid, B Acetonitrile. Gradient: B Conc. 80% - 40% in 0 - 2 min, 40% in 2 - 7 min, 80% in 7 - 12 min, at flow rate of 0.2 mL/min. The elution peak of DNJ was detected by MS using the condition as follow:

Ionization: ESI, Positive mode, Detection: MRM.

The MA powders were extracted with 70% aqueous MeOH and the contents of polyphenols and flavonoids were determined by Folin-Ciocalteu method [21] and Aluminum chloride method [22], respectively.

Briefly, to determine total polyphenols, 50 µL of MA sample extracts were diluted with 2.0 mL of distilled water and reacted with 500 µL of Folin-Ciocalteu’s reagent (Merck KGaA, Germany) and then the absorbance was measured at 750 nm. Gallic acid was used to establish a standard curve and the results were expressed as gallic acid equivalent (mg/g dry sample).

To determine the total flavonoid content, 100 µL of MA sample extracts were reacted with 60 µL of 10% aluminum chloride solution for five minute, following addition of 1 mM sodium hydroxide and 70% ethanol, and then the absorbance was measured at 430 nm. Quercetin was used to establish a standard curve. The results were expressed as quercetin equivalent (mg/g dry sample). All the measurements ware triplicated.

The MeOH extracts of HML and FML were separated on a reverse phase column (phenomenex Gemini C18) equipped with Shimazu HPLC apparatus (LC-10ADvp) by the gradient elution mode with 0.1% formic acid/H₂O and 0.1% formic acid/MeOH as elution solvents. The separated components were monitored by Shimazu SPD-M10A detector at 280 nm absorbance. Chrologenic acid (peak 2) was quantified using authentic sample as standard. Compound A eluted around at Rt = 55 min (peak 12) was qualitatively compared as an example of lipophilic components from the peak hight ratio against Diosmetin used as the internal standard.

Plasma glucose, cholesterol and triglyceride were measured respectively using Glucose C II-test Wako, Cholesterol E-test Wako, and Triglyceride E-test Wako (Wako Pure Chemical Industries Ltd. (Osaka, Japan) according to the protocols attached, respectively. Plasma insulin was determined using LBIS Rat Insulin ELISA Kit (FJIFILM Wako Shibayagi Corporation, Gunma, Japan).

Liver lipids were extracted according to the method by Folch et al. [24]. Briefly, the liver was homogenized in AcOEt to extract lipids. After the solvent was evaporated off, the residual lipids were dissolved in 200 µL of 10% Triton-X 100/isopropanol, and measured triglycerides and total cholesterol as above.

Fixed liver samples were dehydrated using increased gradients of ethanol, embedded in paraffin, and then sectioned into 5 µm thick slices. The tissue slices were stained with Hematoxylin-eosin by standard procedure.

All the data were expressed as mean ± SEM, and analyzed by Statcel 4 software (OMS Publishing Inc., Saitama, Japan). Statistical analysis was performed using the unpaired Student’s t-test to mean of two groups, and one-way ANOVA followed by the Tukey-Kramer HSD test applied for comparisons between multiple experimental groups. Differences were considered statistically significant a p value < 0.05.

The hydroponic cultivation of Morus alba L. (HM) apparently resulted in changing the texture and taste of leaves such that the texture is enough soft and edible as a vegetable. Supporting this, the histological examination revealed the air boundary layer or cuticle of HM leaf looks thiner than FM. The spongy layer also is less stuffed compared to that of FM (Figure 2).

The contents of 1-deoxynojirimycin (1-DNJ), polyphenols were determined for the leaf (HML) and root (HMR) from M. alba cultured by the in-room hydroponics for 3 months and compared to those of field cultivars of MA. Since it is

difficult to obtain the same fibrous root from FM, we could not obtain the data of 1-DNJ contents as well as polyphenols and flavonoids for FMR, and thus they were compared to So-Haku-Hi (mulberry root bark or Sang-Bai-Pi in Chinese) obtained from a Pharmacy store nearby, instead (Figure 3).

It was found that the 1-DNJ contents both in the leaf (HML) of hydroponic cultivar of M. alba and the leaf containing stem (HMLS) samples were apparently high compared to the leaves (FML) obtained from any of 3 different field cultivars examined. The contents in the root of HM are apparently higher than the leaf and also higher than So-Haku-Hi which level is almost same as HML.

On the other hand, the contents of total polyphenol and flavonoid (not shown) in HML and HMLS were almost half of the levels of FML found in the three different field cultivars of MA (Figure 4). However, the polyphenols content of HMR was apparently high compared to any of FMLs and also So-Haku-Hi.

Although the data are not shown, there was a little difference in the polyphenols and flavonoids contents between HML and HMLS such that flavonoids content was higher in HMLS than HML.

HPLC profiling of components in 70% MeOH extracts of HML and FML showed marked difference (Figure 5). The relative distribution of rather hydrophilic components was high in FLM but in contrast, lipophilic components were rich in HML. This is further exampled by comparing the relative amounts of chrologenic acid as typical hydrophilic polyphenols and compound A as an example of

lipophilic polyphenols, respectively, in HML, HMLS and FML. Since the total contents of polyphenols are almost same, it is indicated the lipophilic polyphenols were relatively increased in HML.

The polyphenols contents and DPPH radical scavenging activity was measured in the different solvent extracts of HMR (Figure 6). Since many lipophilic polyphenols are identified as the characteristic ingredients of Morus [12] [25], the root powder was extracted with three different solvents, MeOH, AcOEt and H₂O, and compared the polarity of existing polyphenols. The result showed that the highest distribution of polyphenols was found in the ButOH fraction followed by MetOH and H₂O fractions. So-Haku-Hi also showed the same distribution profile but the content of lipophilic phenols was remarkably low compared to HM in all solvent fractions.

When DPPH radical scavenging activity was measured for each solvent extracted fraction, the highest activity was found in the ButOH extract both for HM and So-Haku-Hi. The DPPH scavenging activity was fairly correlated to the polyphenols content in these extracts from HMR, but the correlation was weak in the case of Ho-Haku-Hi, indicating the components related to the radical scavenging activity are different between HMR and So-Haku-Hi.

Our current studies on component analysis revealed that the relative contents of such polyphenols like oxyresveratrol, Morcin, and Kuwanons are markedly increased in the root as same as in the leaf shown in Figure 4, compared to FM and So-Hakku-Hi (to be published elsewhere).

The body weight changes of rats were followed for 15 weeks by feeding the high fat high sucrose diet (HFHS) with and without supplementation of HML and HMR powders in Figure 7. HFHS fed group showed greater body weight gain compared to the control group fed normal NAI diet during the feeding trial.

However, the body weight gain was significantly suppressed by the supplementation of HMR powder to almost the same level as control group. The HML group also suppressed the body weight gain as well but the extent was small and not significant.

The weekly sum of diet intake during the feeding trial showed no differences between HML and HMR groups, although their levels were significantly low compared to control diet group (data not shown). When the dietary intake volume was compared on the same total calorie base, no significant difference was observed among HFHS, HML and HMR groups, although they were higher than control diet group.

Fasting blood sugar and insulin levels in the plasma after 15 weeks feeding trial were measured (Figure 8). The blood sugar levels did not show significant differences among all test groups, but the insulin levels showed clear differences among them, such that the plasma insulin level was significantly high in HFHS group compared to normal diet control indicating the insulin resistance was acquired, but the insulin level was significantly decreased in both HML and HMR groups, especially in HMR group.

Marked differences in visceral fat volumes after 15 weeks feeding trial were observe among the different diet groups. (Figure 9) The fat volumes of epididymal, perirenal and mesenteric adipose tissues were significantly increased in the rats

fed HFHS but the increases were significantly inhibited in both HML and HMR groups. As same as the effect on the insulin resistance, HMR showed much stronger suppressive effects than HML on mesenteric (p < 0.01), and epididymal (p < 0.01) and perirenal (p < 0.05) fat deposit.

In accordance with the increase of visceral fat volumes, histological observation indicated fatty liver pathogenesis in HFHS diet group after 15 weeks feeding trial. However, in HML and HMR supplemented diet groups, especially HMR group, the liver fat accumulation was apparently reduced and the parenchymal tissue images were significantly ameliorated (Figure 10).

Consistently, both Cholesterol and triglycerides content in the liver was markedly increased in HFHS group, but the increased accumulation of the neutral lipids was significantly decreased in HMR group. HML, however, did not give rise to significant effects on the liver fat accumulation (Figure 11).