Novel Methods to Generate Active Ingredients-Enriched Ashwagandha Leaves and Extracts

Ashwagandha (Withania somnifera) is an Ayurvedic herb commonly used in world-renowned traditional Indian home medicine system. Roots of Ashwagandha have been traditionally known to possess a variety of therapeutic and health promoting potentials that have not been sufficiently supported by laboratory studies. Nevertheless, most, if not all, of the preventive and therapeutic potentials have been assigned to its bioactive components, steroidal alkaloids and lactones. In contrast to the traditional use of roots, we have been exploring bioactivities in leaves of Ashwagandha. Here, we report that the leaves possess higher content of active Withanolides, Withaferin-A (Wi-A) and Withanone (Wi-N), as compared to the roots. We also established, for the first time, hydroponic cultivation of Ashwagandha and investigated the effect of various cultivation conditions on the content of Wi-A and Wi-N by chemical analysis and bioassays. We report that the Withanone/Withaferin A-rich leaves could be obtained by manipulating light condition during hydroponic cultivation. Furthermore, we recruited cyclodextrins to prepare extracts with desired ratio of Wi-N and Wi-A. Hydroponically grown Ashwagandha and its extracts with high ratio of withanolides are valuable for cancer treatment.


Introduction
Plant extracts or their active ingredients constitute about ¼ th of the medicinal drugs. Herbal medicines have not only been known for their safety and efficacy, but also for their affordability and availability to the human populations that either do not have access to the modern medicine or can not afford them. According to World Health Organization (WHO), more than 80% of population in developing countries still relies primarily on traditional herbal

Ethics statement
All in vivo experiments were performed in accordance with the regulations and approval (Experimental Plan Approval #2013-025) of Animal Experiment Committee, Safety and Environment Management Division of National Institute of Advanced Industrial Science & Technology (AIST), Japan.

Preparation of crude alcoholic extract of Ashwagandha leaves
Crude alcoholic extracts of roots and leaves were prepared for chemical analysis. Briefly, dried roots or leaf powder was suspended in 85% ethanol in a ratio of 1:30 and incubated at 85˚C for 2 h in a reflux system. The collected extract was filtered and concentrated by evaporation at 60˚C. The filtrate was lyophilized, by freeze-drying, for overnight. HPLC analysis of the extract was performed using Shimadzu HPLC system (LC-2010A) using YMC-Pack ODS-A (250 × 4.6 mm, 5 μm) column. Purified and well characterized Withaferin A and Withanone were used as standards.

In vitro cytotoxicity assay
Human normal fibroblasts (TIG-3) were procured from Health Science Research Resources Bank, Japan. Osteosarcoma (U2OS) and Fibrosarcoma (HT1080) cell lines were obtained from DS Pharma, Japan, and cultured in Dulbecco's Modified Eagle's Medium DMEM (Invitrogen)-supplemented with 10% fetal bovine serum in a humidified incubator (37˚C and 5% CO 2 ). Cells grown at 40-60% confluency were treated with different kinds of extracts as indicated. Cells were incubated at 37˚C for 48 h following which cytotoxicity assay was estimated by MTT {3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide} assay (Life Technologies) as described earlier [26]. For long-term cell viability, colony-forming assays were performed. One thousand cells were plated in 6-well plate, and cultured either in control or extract-supplemented medium. Cells were cultured for 8-10 days (when colonies appeared in control cultures) with regular change in medium on every alternate day.

Hydroponic cultivation of Ashwagandha
Hydroponic cultivation was established in plant factory that consisted of culture chambers made of (i) heat-insulated panels for temperature-control, (ii) high precision air conditioner and air duct system for airflow and (iii) automated ultrasonic humidifier for moisture control (S1A Fig). The set-up was designed to supply temperature-controlled air into the culture chambers. CO 2 concentration was monitored in the chambers with the help of sensors and was regulated by supplying liquid CO 2 . Culture medium (liquid) for plants, stored in the container on the lowest rack in the culture chamber, was supplied from watering system (circulating pump) that adjusted the ingredients in an automated manner. Alternatively, pretreated seeds were sown in rockwool granule. Four weeks after seeding, the transplants were transferred to hydroponic system under 27/22˚C or 25˚C (as indicated) in light/dark period with 16 h light period and grown for 6 weeks. Stress treatments were started one-two weeks (as indicated) before harvest (Table 1).
Culture racks. Outline of culture racks is shown in (S1B Fig). Culture fluid was supplied to plants periodically by circulation pump (on/off regulated by a timer). Plants were settled in container (pot) filled with culture medium (rockwool granule procured from Nippon Rockwool Corporation, commonly used for strawberry).
Nutrient solution (irrigation system). Adjustment of nutrient solution was done in the ingredient-mixing tank (100 L). Electric conductivity (EC) and pH were controlled to the default settings. Fertilizer A and Fertilizer B were used to make nutrient solution. Herein, liquid fertilizer by OAT-Agrio (A type: OAT-House #1 and #2) was used just as in most plant factories. Concentration of the nutrient solution was adjusted according to EC and the actual plant culture was done using 1 to 1/4 unit of standard concentration, depending on the growing speed (1 unit corresponds to ca 2.4 dS/m EC, while 1/2 unit, ca 1.2 dS/m EC). Nutrient solution adjusted was supplied to circulation tank (25 L) of each rack by supplying pump from the ingredient-mixing tank. The culture fluid supplied to culture beds (which consist of ten or more pots) was recycled in order not to perturb environment. Levels of the nutrient solutions in tanks was controlled by electrodes, and if the nutrient solution became less due to absorption by plants, nutrient solution was supplied automatically from the ingredient mixing tank.
Illumination. Hybrid Electrode Fluorescent Lamp (HEFL) illumination (Nippon Advanced Agri Co. Ltd.) was used to supply four different light wavelengths (i) fluorescent, (ii) Red, (iii) Red:Blue -1:1 and (iv) Red:Blue:Green-1:1:1 (S1C Fig). HEFL emits light with the same principle as fluorescent bulb, and uses exterior electrode tube that is used for large liquid crystal screen TV. Energy consumption is lower as compared to other illumination methods. Since heat generation is also low, close illumination to the plants was possible. It was also suitable for growing plants in a multiple layers and offered large cultivation area [36].

Flow of culture experiments
Culture experiments were performed according to the flow shown in S1D Fig. Seeds were sown after pre-treatment that consisted of cleaning with water and treatment with gibberellin and low temperature. After about one week, when germination took place, seedlings were grown for about three weeks in culture fluid and transplanted on culture rack in culture chamber. Thus the hydroponic cultivation was started. Two weeks post-transplantation, nutrient solution (one unit of standard concentration) was supplied, and hydroponic culture was continued. The plants were harvested two months post-transplantation. Temperature of the culture chamber was adjusted to 25˚C both for light (16 h) and dark (8 h) periods. CO 2 concentration in the light period was controlled to ca. 1000 ppm ( Table 1).

Preparation of cyclodextrin-assisted water extract of Ashwagandha leaves
Water extract (10% w/v) was prepared from Ashwagandha dried leave powder, as described earlier, by overnight extraction in sterile water at 40˚C with slow shaking [25]. For cyclodextrin (CD)-assisted aqueous extraction of Ashwagandha leaves (CD-WEX), the dried leaf powder (10% w/v) was mixed with aqueous solution of alpha (10%) or beta (2%) or gamma (10%) CD. The mixture was stirred for 24 h at 37˚C with slow shaking (90 rpm) in TAITEC Bio-Shaker BR-43FL. The slurry was centrifuged at 3500 rpm for 10 min and the supernatant was filtered through 0.45-micron filter. The filtrate (CD extract) was subjected to HPLC and bioassays. High Withanone: Withaferin A ratio was identified in the residual precipitate of gamma CD extraction. In order to investigate anticancer potential of these extracts, the active components were extracted from precipitate in DMSO as described above. The supernatant (DM extract) obtained after centrifugation at 3500 rpm for 10 min was filtered through 0.45-micron filter and used for cytotoxicity assays. CD and DM extracts, obtained from dry leaf powder (10% by weight), were considered 100% and added to the cell culture medium in a range of 0.01~1% that corresponded to 10 μg~1 mg/ml of leaf powder, respectively.

High-pressure liquid chromatography (HPLC)
The HPLC for alpha, beta and gamma CD-assisted water extract of Ashwagandha (WEX) preparations and the gamma CD-complex was performed using

In vivo tumor formation assays
Balb/c nude mice (4 weeks old, female) were bought from Nihon Clea (Japan). Animals used for experimentation received human care. All in vivo experiments were performed in accordance with the institutional regulations as approved by animal experiment ethical committee. Mice were housed under pathogen free conditions under a 12 h dark/light cycle and fed with standard chow ad libitum. For anti-tumor assays, HT1080 cells (6 x 10 6 cells suspended in 0.2 ml of growth medium) were injected into the nude mice subcutaneously (two sites per mouse). Control group was treated with 2% carboxymethyl cellulose (CMC). WEX group was fed with 500 mg WEX/Kg body weight and CD-WEX group was fed with 500 mg WEX and 0.625 mg gamma CD/Kg body weight. The treatment started on the 8th day post-injection of cells, and was carried out 12 times on alternate days. Tumor formation was monitored for a month. Predefined human endpoints were established according to National Institute of Advanced Industrial Science & Technology (AIST), Japan Committee on the Ethics of Animal Experiments. Criteria set for need to euthanize was the tumor size, physical appearance including sickness, distress or immobility. Maximum tumor size allowed was 20 mm at the largest diameter. None of the animals, in the present study, met any criteria that required euthanization. The volume of subcutaneous tumors was calculated as V = L X W 2 /2, where L was length and W was width of the tumor, respectively. For metastasis assay, the recipient mice were sacrificed by cervical dislocation, lungs were fixed in 4% formaldehyde and the tumor colonies were counted 5 weeks after tail vein injection. This assay was performed using three mice for each group, and repeated twice.

Results and Discussion
Alcoholic extract of Ashwagandha leaves has been shown to possess anticancer activity in in vitro and in vivo assays [25][26][27][28]37]. Withanolide constituents present in the alcoholic extract of leaves, such as Withanone and Withaferin A, were shown to kill cancer cells by mechanisms involving apoptosis and growth arrest [25][26][27][28][38][39][40][41][42][43][44]. Whereas Withanone causes selective cancer cell killing, Withaferin A, at high concentration, was seen to possess cytotoxicity to normal cells in in vitro assays. Addition of Withanone along with Withaferin A to the culture medium protected the normal cells against cytotoxicity of the latter [26]. Several NMR studies have shown that the Ashwagandha leaves collected from either different origins or different stages of development vary in the ratio of the Withanolides [45][46][47][48]. In order to explore the use of Ashwagadha for cancer treatment, we investigated the content of Wi-A and Wi-N in plants cultivated in Ibaraki and Tokushima (Japan) and Punjab (India). Comparative HPLC analyses of the alcoholic extracts of leaves and roots of these plants were carried out. The analyses revealed that the yield of Withanolides was several fold higher in leaves as compared to the roots raised at distant places (Fig 1A and 1B and S1E Fig). Cytotoxicity of these extracts for human cancer cells was investigated by short term and long term cell viability assays. Consistent with the high content of Withanolides in leaves, the leaf extracts showed higher toxicity to human cancer cells as compared to the root extracts ( Fig 1C). Variations in chemotypes of Ashwagandha have been an issue for its use and value as a medicinal herb [45,47]. We envisaged that the hydroponic cultivation might be useful to provide uniform resource of bioactive Ashwagandha. In the present study, we demonstrate, for the first time, hydroponically grown Ashwagandha and evaluation of its leaves for anticancer bioactives. As shown in Fig 2A and S1 Fig, hydroponic system was set up. Under the conditions described in Material and Methods Section and S1 Fig, we successfully obtained hydroponically grown Ashwagandha, as shown in Fig 2A and 2B, and S1 Video. Furthermore, we investigated the effect of a variety of environmental conditions including, exposure to UV, temperature, pH and nutrients (Table 1). Plants grown under different conditions were examined for various attributes including, plant height, number of leaves, weight of shoots and roots (Fig 2C and 2D). Such analyses revealed that Ashwagandha is tolerant to a variety of stress conditions and did not show any dramatic changes in several plant attributes (Fig 2C). Some conditions that caused noticeable changes in root and leave attributes included (i) cultivation in four units of nutrient solution caused hypertrophic roots (Fig 2D), (ii) cultivation in nutrient solution containing four units of NaCl caused decrease in root mass (Fig 2D). Hydroponic plants cultivated in stressed conditions including exposure to UV and high temperature showed some visible alteration in growth. UV-B exposure for over 30 min/day caused dramatic leaf damage and death of plants. Therefore, UV-B exposure was restricted to 10 min. We noticed that the plants exposed to UV-B during night caused curling of leaves (Fig 2E). UV-A (16 h, during light period) exposure was well tolerated by the plants. Biological activity analysis of the leaf extracts from UV-stressed plants showed high toxicity to cancer cells suggesting that in spite of the above described phenotypic changes observed in the stressed plants, there was no major impact on anticancer bioactives. Similarly, we investigated the effect of high temperature stress. Light/dark (42/22˚C) was lethal, (37/22˚C) yielded shorter plants with more lateral shoot branching that possessed thick and dense green leaves. Similar to the UV-stressed, these plants also showed no difference in their cytotoxic activity in the extracts as compared to the control plants (Fig 2F). Leaves of hydroponically grown plants, under a variety of environmental conditions, were also examined for the content of Withaferin A and Withanone (Fig 3A). We found that similar to the leaves from plants cultivated on land, hydroponically grown plant leaves possessed higher content of Withanone than Withaferin A under all environmental conditions. Human cancer cells cytotoxicity assays revealed that the hydroponically grown leaves also possess anticancer activity. Furthermore, higher toxicity to cancer than to the normal cells was consistent with high Withanone content in the hydroponically grown leaves (Fig 3B).
We next investigated the effect of different light conditions on hydroponically grown plants (S1C Fig (Fig 4A). Similar to the plants raised on land, the root extract of hydroponically raised plants possessed less withanolides than the leaves (Fig 4A), and showed low cytotoxicity to cancer cells (Fig 4B). Furthermore, we found that the leaf extract from plants raised under red light had 10-fold higher Withanone than Withaferin A, and were highly cytotoxic to cancer cells (Fig 4C).
In contrast to the alcoholic extract, water extract is more favorable due to the ease in preparation and compatibility with human food. Hence, the activities in the water extract were investigated [25]. We demonstrated that the water extract of Ashwagandha leaves possess anticancer activity. The active anticancer component was identified as triethylene glycol (TEG), in addition to low level of Withaferin A and Withanone, by chemical characterization including HPLC and NMR. These results predicted the need of novel extraction method(s) to obtain a mixture of alcohol-and water-soluble compounds in a moderate level for superior anticancer effects. In light of the above information, we investigated the potential of various isoforms of cyclodextrin (CD) for the preparation of aqueous anticancer extracts from Ashwagandha leaves. CDs are natural derivatives of starch or polymer of glucose that possess circular structure. These are widely used in food, pharmaceutical, agriculture, and environmental engineering and drug delivery because of its structure (hydrophobic inside and a hydrophilic outside) that enhances the solubility and bioavailability of compounds. Accordingly, aqueous extractions of hydrophobic drugs and health ingredients from plant materials by using CDs have been reported [49][50][51]. Gamma CD, widely accepted as food constituent, consists of 8 glucose monomers arranged in the form of a cyclic ring. It has been reported to enhance the bioavailability of hydrophobic ingredients such as coenzyme Q10 [52][53][54]. We recruited CDs for aqueous extraction of bioactives from Ashwagandha leaves. As shown in Fig 5A, we found that the CD-assisted aqueous extraction of Ashwagandha leaves resulted in an enrichment of anticancer Withanolides. Beta CDderived extracts of Ashwagandha leaves contained highest level of Withanone and Withaferin A. By cell-based assays, we found that the CD extracts of Ashwagandha leaves have enhanced cancer cell cytotoxicity as compared to the conventional water extract. HPLC analysis of Withanone and Withaferin A in gamma-CD residual precipitate revealed that they contained 17-fold higher Withanone than Withaferin A (Fig 5B). We earlier reported that Withanone:Withaferin A (in the ratio of 20:1) possess high anticancer and anti-metastasis activities [14] and hence hypothesized that the gamma-CD residual precipitates could be very useful for cancer treatment. We validated by in vitro and in vivo experiments. For cell culture experiments, the bioactives in gamma-CD residual precipitate were extracted in DMSO (called DM extracts). Cell-based viability assays revealed that the CD extracts were more cytotoxic to both cancer and normal cells. Root extracts that contained low level of Withaferin A and Withanone were ineffective (Fig 5C). DM extracts showed higher cytotoxicity to cancer, and milder to normal, cells. Quantitative MTT assays endorsed that whereas root extracts (both CD and DM, with low level of Withanolides in each) had IC50 >2% for cancer and normal cells, the leaf-CD extracts were cytotoxic to both cancer (IC50~0.1%) and normal (IC50~1%) cells. Leaf-DM extracts showed higher toxicity to cancer cells (IC50 0.125% as compared to >2% for normal cells) (Fig 5D). The results were confirmed by extracts generated from hydroponically cultivated leaves exposed to (i) red light-high Withanone (S-C2) and (ii) UV-high Withaferin A (S-A2) (Fig 2). S-C2DM extracts from hydroponically cultivated plants showed selective toxicity to cancer cells. In in vivo nude mice tumor formation assays, aqueous extract of leaves and gamma CD combination caused stronger suppression of subcutaneous tumors and lung metastasis in nude mice (Fig 5E and  5F). Taken together, it is strongly suggestive that cyclodextrins are useful for aqueous extraction of bioactives in Ashwagandha leaves that could significantly enhance the anticancer activity in vivo.

Conclusion
We have succeeded in establishing a hydroponic cultivation of Ashwagandha with enriched bioactives. We demonstrate that the leaves of Ashwagandha possess high content of bioactives and it could be further manipulated by light conditions during their cultivation. Whereas red light yielded leaves with high content of Withanone, UV light resulted in high level of Withaferin A. Furthermore, we have developed a new method of extraction for preparing Withanone-rich extracts that could be used in effective cancer treatment.