Kamal Ram Arya

A novel flavonoid isolated from the steam-bark of Ulmus wallichiana planchon stimulates osteoblast function and inhibits osteoclast and adipocyte differentiation.

(2S,3S)-Aromadendrin-6-C-β-d-glucopyranoside (AG) is a novel flavonol isolated from the extract of Ulmus wallichiana (Himalayan Elm). Extract of U. wallichiana is used as a traditional medicine for rapid fracture repair in India. We characterized the mechanism of action of AG in mouse bone cells by investigating its effect on the precursors of osteoblasts, osteoclasts and adipocytes. At nanomolar concentrations, AG increased differentiation of preosteoblasts obtained from neonatal mouse calvaria. The gene expression of osteogenic markers, including runt-related transcription factor 2 (Runx-2), bone morphogenetic protein-2 (BMP-2), type I collagen and osteocalcin were elevated in the preosteoblasts. The extracellular matrix mineralization was higher in preosteoblast and bone marrow cells when AG was present in the medium. Furthermore, AG protected the differentiated osteoblasts from serum deprivation-induced apoptosis, and increased the expression of the anti-osteoclastogenic cytokine, osteoprotegerin. It inhibited osteoclast differentiation of bone marrow precursor cells to osteoclasts in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and monocyte/macrophage-colony stimulating factor (M-CSF). Additionally, in 3T3-L1 preadipocytes, AG decreased the expression of genes involved in adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein (SREBP) and CCAAT/enhancer-binding protein alpha (CEBP/α), and induced apoptosis of differentiated adipocytes. Induction of adipogenic differentiation was also inhibited in the presence of AG. AG exhibited no estrogenic/antiestrogenic effect. Together, our data show that AG has potent osteogenic, anti-osteoclastogenic and anti-adipogenic effects, which may translate to a better skeletal outcome in postmenopausal osteoporosis.

Extract and fraction from Ulmus wallichiana Planchon promote peak bone achievement and have a nonestrogenic osteoprotective effect.

Objective: This study aimed to determine the skeletal effects of total ethanolic extract (TEE) and its butanolic fraction (BF) from the stem-bark of Ulmus wallichiana, which is rich in C-glycosylated flavonoids, in growing rats (for peak bone [PB] achievement) and in ovariectomized (OVx) rats (for menopausal bone loss). Methods: TEE (750 mg kg−1 d−1) and BF (50 mg kg−1 d−1) were given orally for 10 weeks to weaning female Sprague-Dawley rats and for 12 weeks to adult OVx rats of the same strain, respectively. In studies with OVx rats, sham operated + vehicle, OVx + 17&bgr;-estradiol, and OVx + vehicle groups served as various controls. Bone mineral density (BMD), biomechanical strength, bone histology, formations of osteoprogenitor cells, osteoid formation, and bone turnover/resorption markers were studied. Bioactive marker compounds in TEE and BF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test significance of effects. Results: In growing rats, both TEE and BF increased BMD, bone strength, and bone formation rate, suggesting higher PB achievement. OVx rats treated with either TEE or BF exhibited increased BMD at various anatomical positions and improved bone strength and trabecular architecture compared with the OVx + vehicle group. Serum osteocalcin and urinary type 1 collagen degradation product levels in OVx rats treated with either TEE or BF were significantly lower than those of the OVx + vehicle group. Neither TEE nor BF exhibited uterine estrogenicity. Analysis of marker compounds revealed significant enrichment of two bioactive markers in BF over TEE. Conclusions: Derived from U wallichiana, BF at much a lower dose than TEE was effective in PB achievement and prevention of OVx-induced bone loss.

Constituents from fruits of Cupressus sempervirens.

Two new phenolic glycosides (1, 2), along with fourteen known compounds (3-16) have been isolated from the fruit of Cupressus sempervirens. The structures of these compounds were determined by spectroscopic analysis and were evaluated for their inhibitory activity against glycogen phosphorylase and glucose-6-phosphatase enzymes. Compounds 14 showed a moderate inhibition against glucose-6-phosphatase and 15 against glycogen phosphorylase enzymes.

In vitro and in vivo anticancer activity of 2-deacetoxytaxinine J and synthesis of novel taxoids and their in vitro anticancer activity

The taxane diterpneoid 2-deacetoxytaxinine J (2-DAT-J) 1 has been isolated from the bark of Himalayan yew, Taxus baccata L. spp. wallichiana in a reasonably good yield (0.1%) and its anticancer activity against breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human kidney epithelial cell line (HEK-293) has been studied. 2-DAT-J (1) showed significant in vitro activity against breast cancer cell line at a concentration of 20 microM and 10 microM in MCF-7 and MDA-MB-231 respectively. Few novel taxoids were derived (7, 8 and 10-13) from the naturally occurring 2-DAT-J (1) and screened for their anticancer activity. The structure-activity relationship studies indicated that the cinnamoyl group on C-5 and acetyl group on C-10 are essential for the anticancer activity. 2-DAT-J (1) was also tested for its in vivo activity on DMBA-induced mammary tumors in virgin female Sprague Dawley rats at a dose of 10mg/kg body weight orally for 30 days and showed significant regression in mammary tumors as compared to vehicle treated group (p<0.05).

Rapid profiling and structural characterization of bioactive compounds and their distribution in different parts of Berberis petiolaris Wall. ex G. Don applying hyphenated mass spectrometric techniques

RATIONALE Berberis petiolaris Wall. is a lesser known medicinal plant, belonging to the family Berberidaceae. The genus Berberis is known for many biological activities such as anti-microbial, anti-inflammatory and anti-diarrheal, etc. There are not many reports of the isolation of components from Berberis petiolaris. This study aims to seek identification, characterization and quantification of components. METHODS A method was developed for rapid screening of phytochemicals using high-pressure liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry (HPLC/ESI-QTOF-MS/MS). Suitable collision-induced dissociation mass spectrometry (CID-MS/MS) methods were developed for structural investigation of alkaloids, flavanoids and other classes of compounds using nine reference standards for authentication. Multiple reaction monitoring (MRM) methods were developed for quantitative study of five constituents using triple quadrupole-linear ion trap mass spectrometry (UPLC/QqLIT-MS/MS). RESULTS On the basis of HPLC retention behavior and fragmentation pathways obtained by high-resolution MS and MS/MS, 32 compounds were identified and characterized in different parts of Berberis petiolaris. Quantitative studies of chlorogenic acid, magnoflorine, jatrorrhizine, palmatine and berberine were also completed successfully. CONCLUSIONS Rapid and accurate HPLC/ESI-QTOF-MS/MS and UPLC/ESI-QqLIT-MS/MS methods were established for identification, characterization and quantification of phytochemicals in the ethanolic extract of Berberis petiolaris. These methods, therefore, can be used for studies on phytochemical variation in different parts of the plant. Principle components analysis (PCA) may be used for plant part discrimination.

Anti-breast tumor activity of Eclipta extract in-vitro and in-vivo: novel evidence of endoplasmic reticulum specific localization of Hsp60 during apoptosis

Major challenges for current therapeutic strategies against breast cancer are associated with drug-induced toxicities. Considering the immense potential of bioactive phytochemicals to deliver non-toxic, efficient anti-cancer therapeutics, we performed bio-guided fractionation of Eclipta alba extract and discovered that particularly the chloroform fraction of Eclipta alba (CFEA) is selectively inducing cytotoxicity to breast cancer cells over non-tumorigenic breast epithelial cells. Our unbiased mechanistic hunt revealed that CFEA specifically activates the intrinsic apoptotic pathway by disrupting the mitochondrial membrane potential, upregulating Hsp60 and downregulating the expression of anti-apoptotic protein XIAP. By utilizing Hsp60 specific siRNA, we identified a novel pro-apoptotic role of Hsp60 and uncovered that following CFEA treatment, upregulated Hsp60 is localized in the endoplasmic reticulum (ER). To our knowledge, this is the first evidence of ER specific localization of Hsp60 during cancer cell apoptosis. Further, our LC-MS approach identified that luteolin is mainly attributed for its anti-cancer activities. Moreover, oral administration of CFEA not only offers potential anti-breast cancer effects in-vivo but also mitigates tumor induced hepato-renal toxicity. Together, our studies offer novel mechanistic insight into the CFEA mediated inhibition of breast cancer and may potentially open up new avenues for further translational research.

Rapid screening for the adulterants of Berberis aristata using direct analysis in real-time mass spectrometry and principal component analysis for discrimination

Adulteration or substitution of commercial Berberis aristata and its herbal products with inferior-quality substituents is very common. Metabolic profiling of B. aristata, along with its common adulterants/contaminants/substituents such as B. asiatica, Mahonia borealis and Coscinium fenestratum, was rapidly carried out using direct analysis in real-time mass spectrometry (DART MS) to generate the chemical fingerprints for the differentiation of these species. Phytochemical analysis showed the presence of mainly alkaloids. The identified alkaloids were berberrubine, berberine, jatrorrhizine, ketoberberine, palmatine, dihydropalmatine or 7,8-dihydro-8-hydroxyberberine, berbamine and pakistanamine. Berberine, which was mainly reported from the root and stem bark of B. aristata, was also identified in the leaf along with chlorogenic acid. The DART MS data have been subjected to principal component analysis (PCA). The resulting score plots showed clustering and clear differentiation of the species and plant parts. It is thus apparent that the technique of DART MS followed by PCA is a quick and reliable method for the direct profiling of B. aristata and its adulterant plants and plant parts. The study reports the rapid analytical method to identify the possibility of illegal adulteration/contamination/substitution in potential plant materials and herbal extracts. Graphical Abstract

Quantitative determination of isoquinoline alkaloids and chlorogenic acid in Berberis species using ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry

Berberis species are well known and used extensively as medicinal plants in traditional medicine. They have many medicinal values attributable to the presence of alkaloids having different pharmacological activities. In this study, a method was developed and validated as per international conference on harmonization guidelines using ultra high performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry operated in the multiple reaction monitoring mode for nine bioactive compounds, including protoberberine alkaloids, aporphine alkaloids and chlorogenic acid. This method was applied in different plant parts of eight Berberis species to determine variations in content of nine bioactive compounds. The separation was achieved on an ACQUITY UPLC CSH™ C18 column using a gradient mobile phase at flow rate 0.3 mL/min. Calibration curves for all the nine analytes provided optimum linear detector response (with R(2) ≥0.9989) over the concentration range of 0.5-1000 ng/mL. The precision and accuracy were within RSDs ≤2.4 and ≤2.3%, respectively. The results indicated significant variation in the total contents of the nine compounds in Berberis species.

Saraca indica Bark Extract Shows In Vitro Antioxidant, Antibreast Cancer Activity and Does Not Exhibit Toxicological Effects

Medicinal plants are used as a complementary and alternative medicine in treatment of various diseases including cancer worldwide, because of their ease of accessibility and cost effectiveness. Multicomposed mixture of compounds present in a plant extract has synergistic activity, increases the therapeutic potential many folds, compensates toxicity, and increases bioavailability. Saraca indica (family Caesalpiniaceae) is one of the most ancient sacred plants with medicinal properties, exhibiting a number of pharmacological effects. Antioxidant, antibreast cancer activity and toxicological evaluation of Saraca indica bark extract (SIE) were carried out in the present study. The results of the study indicated that this herbal preparation has antioxidant and antibreast cancer activity. Toxicological studies suggest that SIE is safer to use and may have a potential to be used as complementary and alternative medicine for breast cancer therapy.

Labda‐8(17),12,14‐trien‐19‐oic Acid Contained in Fruits of Cupressus sempervirens Suppresses Benign Prostatic Hyperplasia in Rat and In Vitro Human Models Through Inhibition of Androgen and STAT‐3 Signaling

Fruit extract of Cupressus sempervirens (CS), which is used traditionally to treat Benign Prostatic Hyperplasia (BPH)‐like urinary symptoms in patients, was scientifically validated for anti‐BPH activity. The ethanolic fruit extract of CS inhibited proliferation of human BPH‐stromal cells and the activity was localized to its chloroform‐soluble, diterpene‐rich fraction. Eight major diterpenes isolated from this fraction exhibited moderate to potent activity and the most active diterpene (labda‐8(17),12,14‐trien‐19‐oic acid) exhibited an IC50 of 37.5 μM (antiproliferative activity against human BPH‐stromal cells). It significantly inhibited activation (phosphorylation) of Stat‐3 in BPH‐stromal cells and prevented transactivation of androgen sensitive KLK3/PSA and TMPRSS2 genes in LNCaP cells. Labda‐8(17),12,14‐trien‐19‐oic acid‐rich CS fraction prevented prostatic hyperplasia in rat model and caused TUNEL labeling of stromal cells with lower expressions of IGF‐I, TGF‐ß and PCNA, and bcl‐2/bax ratio. Human BPH tissues exhibited precise lowering of stromal component after incubation in labda‐8(17),12,14‐trien‐19‐oic acid, ex vivo. We conclude that labda‐8(17),12,14‐trien‐19‐oic acid contained in CS exhibits anti‐BPH activity through inhibition of stromal proliferation and suppression of androgen action in the prostate, presenting a unique lead structure for further optimization of anti‐BPH activity. Copyright