This investigation includes for the first time, as part of an ongoing research, a new chemical compound and main constituents isolated from P. nestorianus and bioassay evaluation. In fact, the finding of new chemical compounds from medicinal plants has now become the main goal of several researchers, due to their medical and pharmacological importance.

Several techniques were performed for gross separation, further purification and fractionation such as extraction by maceration, solid phase extraction (SPE), liquid-liquid extraction, thin layer chromatography (TLC), column chromatography (CC) on silica gel, reversed phase (RP-18) and sephadex-LH20, steam distillation and medium performance liquid chromatography (MPLC). The structures were characterized and identified using spectroscopic techniques, in particular 1H- and 13C- 1D and 2D-NMR (COSY, NOESY, HMBC and HSQC) and LC-ESI-MS.

The most significant results of this study are the identification of a new iridoid dimer, along with seven known compounds possessing rare structures. They include  three flavonoid  derivatives, luteolin-7-O-glucoside, kaempferol 3-(3”-acetyl-6”-p-coumaroylglucoside) and kaempferol 3-(3”, 6”-di-Op-coumaroylglucoside); two phenolic acids, 3-O-caffeoylquinic acid and 3,5 di-O-caffeoylquinic acid; three iridoids, loganic acid and kurdnestorianoside (a new compound) from the flower methanol extract, and loganin from the roots. The presence of flavonoids and caffeoyl derivatives may well support the use of P. nestorianus as an anti-inflammatory remedy in local medicine.

Sixty-five elements have been identified in P. nestorianus using ICP-OES and ICP-MS techniques; Among the main essential elements, the highest concentration was found  for Ca in leaves (36.1 g/kg), followed by K,  Mg, S,  Fe,  Al and P (6.9, 5.53, 3.2 , 1.92 , 1.85 and 0.79 g/kg), respectively. On the other hand, low concentrations of Bi (0.005 mg/kg), Pt (0.002 mg/kg) and Au (0.0007 mg/kg) were determined in flowers, leaves and roots, respectively.

A qualitative phytochemical investigation of different extracts of P. nestorianus flowers, leaves and roots was performed. Of the eight phytochemical tests done, six were positive for flavonoids, phenols, terpenoids, saponins, glycosides and steroids. Instead, alkaloids and tannins were absent in all extracts. Moreover, total phenolic content (TPC) and total flavonoid content (TFC) were quantified. TPC values ranged from 61 to 200 mg /g extract, while the TFC values of flower, leaf and root ethyl acetate and methanolic extracts varied from 8 to 80 mg /g extract.

The chemical components of the volatile fractions obtained by hydrodistillation of P. nestorianus flowers and leaves were fully investigated by GC. A total of fifty-five constituents were identified, constituting 99.68% and 99.04% of the total flower (PFO) and leaf (PLO) oils, respectively. The oils were obtained with yields of 0.15% (w/w) and 0.10%, respectively, on dried vegetable material. The prevalent constituents of the PFO were α-terpineol (2.41%), α-linalool (6.42%), 6,10,14-trimethyl-2-pentadecanone (2.59%), myristic acid (24.65 %), and lauric acid (50.44%), while the major components of PLO were (E)-hex-2-enal (2.26%), (E)-hex-2-en-1-ol (2.04), myristic acid (34.03%), and lauric acid (50.35%).

In another part of this investigation, the bioassay activities of extracts, essential oils and isolated compounds were tested for the first time. Thus, the antioxidant, antifungal and antiproliferative properties were determined. In the DPPH assay the extracts of flowers and leaves showed higher antiradical activity than the EtOAc extract of roots, appearing to be as potent as gallic acid. Actually, the maximum inhibitory activity was about 87%, comparable to gallic acid (89.9%), while the EtOAc extract of roots was much less active (29.6%).

Moreover, the two volatile fractions showed significant inhibitory and fungicidal activities against the medically important fungi Candida albicans, C. tropicalis, Microsporum canis, and Trichophyton mentagrophytes, with MICs ranging from 0.7 to 3.3 mg/mL and MFCs varying from 1.4 to 6.6 mg/mL. The antiproliferative activity of the two oils was assayed on several  human tumor cell lines, in particular, MCF7 and SkBr3 breast, IST-MES1 mesothelioma, A549 lung, BG-1 ovarian and Ishikawa endometrial cancer cells and human mammary MCF-10A epithelial cells. Both volatile fractions showed selective antitumor activity, with IC50 ranging from 1 to 3 µg/mL, even higher than that of cisplatin.

In addition, a couple of isolated kaempferol derivatives showed a remarkable antiproliferative activity on several human tumor cell lines, in comparison with the well-known chemotherapeutic agent cis-diamminedichloroplatinum (II) (cisplatin). Notably, kaempferol 3-O-(3′′, 6′′-di-OEp-coumaroyl)-β-D-glucopyranoside revealed an inhibitory activity even higher than that of cisplatin, with IC50 values ranging from 2 to 6 µM.

Finally the results of bioassay activities and those reported in literature for the isolated compounds support and validate the uses of P. nestorianus as medicinal remedy.