A Spotlight on Chemical Constituents and Pharmacological Activities of Nigella glandulifera Freyn et Sint Seeds

Plants belonging to the Ranunculaceae family, and particularly their seeds, have been a hot research topic in numerous pharmacognosy laboratories. Nigella glandulifera Freyn et Sint (NG) is one of the promising, but relatively insufficiently studied, plants from this family. In this review, we summarize the recently isolated chemical constituents from the seeds of this plant including alkaloids, flavonol glycosides, isobenzofuranone derivatives, saponins, terpenes, terpenoids, and fatty acids. We put also a spotlight on the recently studied therapeutic potentials of such amazing herb seeds as antidiabetes, melanogenesis inhibition, anticancer, anti-inflammatory, antithrombosis, and antiplatelet aggregation effects. Herein, we illustrate certain properties and potentials via selected examples, and thus we suggest more studies to confirm the therapeutic hypotheses, find out new compounds, and eventually to discover novel properties.


Introduction
Medicinal plants from the buttercup family (Ranunculaceae) have been applied extensively for many years to treat various diseases, particularly the seeds.The Chinese plant NG is an annual erect herbaceous plant from this family [1].It has been reported that NG seeds have diuretic, spasmolytic, analgesic, galactagogue, and bronchodilator effects.Because of their medicinal effects as a natural product, they have been used for a long time to treat urinary calculus, oedema, and bronchial asthma [2].
For the last three decades, three alkaloids with indazole ring system, namely, nigeglanine, nigellicine, and nigellidine, have been isolated from two plants of this family, that is, Nigella sativa (NS) and NG [3].Indazoles are rare in nature, and these are the only three natural products based on indazole ring being isolated [4].The indazole ring system attracts great interest as partial structure of numerous biologically active compounds [4], hence, the importance of this family in pharmacological research.
Nigella genus consists of about 20 species, including NG, NS, and Nigella damascena, which are used in traditional medicine [5].In southwest and western part of China (Xinjiang autonomous region, Yunnan and Tibet), NG is dis-tributed widely and its seeds have been used since antiquity.NG seeds are mentioned in the Pharmacopoeia of China since 1997 [1,2,6,7].This review aims to put a spotlight on this plant relatively lacking in studies and research compared to other herbs of the same genus, such as NS.We aim also to provide a useful summary of compounds isolated recently from NG seeds and their potential therapeutical properties newly discovered.
NG seeds are rich in various chemical constituents, such as alkaloids [1], flavonol glycosides [8], isobenzofuranone derivatives [9], saponins [9,10] terpenes, terpenoids [11] and fatty acids [6].This plant's seeds are used not only as a spice but also as a treatment of some diseases, such as bronchial asthma, in southwest and western part of China [10].Recently, more and more scientists are trying to test chemical constituents and therapeutical capacities of this herb.This plant seeds contain p-cymene and thujene, which may interfere with dephosphorylation of the insulin receptor and leptin signaling [12], and also oleic acid methyl ester and dioctyl phthalate (DOP) that inhibit the melanogenesis [13,14].They contain also kalopanaxsaponins A and I, which may be potential therapeutic agents to treat the parental and drug resistant hepatoma [2].In addition, NG seeds total saponins have been reported as having an anti-inflammatory effect

Chemical Constituents Isolated from NG Seeds
2.1.Alkaloids.Nigeglanine and its artificial derivative (named Nigeglapine [17]) have been isolated from the seeds of this plant, together with fuzitine (a known aporphine alkaloid) [1].Nigeglanine is based on indazole ring system, and this structure is seldom found in natural compounds [4].Moreover, the indazole ring system is very interesting as partial structure of many bioactive compounds [18].For instance, a novel class of human bradykinin B1 receptor antagonists consist of indazole derivatives [18].A structureactivity relationship examination of the indazole moiety in some compounds was performed en route as participating for an optimal bradykinin B1antagonist [18].The indazole part of molecules studied was identified as a sensitive feature for the optimal binding to the receptor with a chlorine at the 3-position of the indazole optimal for high receptor affinity, and this is interesting in developing selective bradykinin B1 receptor antagonists [18].Hence, the possibility for nigeglanine, in addition to other similar compounds isolated from NG seeds, to provide a starting point for semisynthesis of this kind of interesting compounds since it is one of the rare natural compounds with indazole ring, however this remains to be experimentally proved and investigated.
Most recently, four dolabellane-type diterpene alkaloids were isolated for the first time from NG seeds.They were identified as nigellamine A 1 , nigellamine A 2 , and nigellamine B 1 , in addition to nigeglamine, which was a new natural compound [19].

Flavonoids and Phenolic
-D-glucopyranoside, was isolated from the seeds of NG [8].
The chemical structures of thymoquinone and other selected compounds isolated from NG seeds are shown in Figure 1.

Pharmacological Activities and Related Active Compounds
3.1.Antidiabetes and Antiobesity Properties.Non-insulindependent diabetes mellitus (NIDDM) fundamental feature is resistance to insulin [12].Another hormone, leptin, is secreted by adipose tissues throughout the body and its best known function is the appetite feedback control [23].
Recently, many evidences have shown that protein tyrosine phosphatase 1B (PTP1B) is involved in the downregulation of insulin and in leptin signaling [12].This enzyme is a key element in insulin signaling negative regulation pathway, hence, the importance of its role in diabetes and obesity.
Many studies have shown that PTP1B plays a pivotal role in the dephosphorylation of insulin receptors [24].Hence, it is clear that the inhibition of PTP1B ought to be a potential pharmacological mechanism for the treatment of NIDDM.The potential antidiabetic effect in NG seeds was confirmed based on in vitro enzyme assays for protein tyrosine phosphatase inhibitory activities, and furthermore, the most convincing evidence is that PTP1B was involved in the insulin-signaling pathway deriving from the phenotype of the PTP1B knockout (PTP1B KO) mice [12].These mice (PTP1B−/−) completely deficient in this enzyme showed increasing phosphorylation of insulin receptor, increasing sensitivity to insulin and resistance to diet-induced obesity, and in addition, their growth and development were normal, they were fertile, and were histologically just like wild-type mice (PTP1B+/+) [12,[25][26][27], thus the importance of PTP1B as a drug target for NIDDM and obesity treatment.Moreover, the difference in chemical constitutes and antidiabetes properties of NG oil extracted by n-hexane (HNG) and petroleum ether (PNG) was investigated [12].Gas chromatography-mass spectrometry method was used to analyse their chemical components, and different concentrations of PNG and HNG were assayed for their PTPB1 inhibitory effects [12].The findings showed that, in both of them the unsaturated fatty acids are the main components, there were about 87% in PNG less than them in HNG, and that PNG can inhibit PTP1B with half maximal inhibitory concentration values of 33.15 ± 0.77, against 18.50 ± 0.52 mg/mL for HNG.Thus, it was suggested that not only the unsaturated fatty acids, but also the small molecules compounds, such as p-cymene and thujene, have the predominantly effect in inhibiting PTP1B activity [12].

Skin Depigmenting Properties.
Melanin is a brown, yellow-brown, or black pigment produced by melanocytes.These cells manufacture this phenolic biopolymer from the amino acid tyrosine molecules [28].The melanogenesis is started by the hydroxylation of tyrosine into DOPA, then oxidation of DOPA to dopaquinone; interestingly, this first and rate-limiting step of melanin formation is mediated by tyrosinase, which is the key enzyme required for melanin production [29,30].After that, dopaquinone undergoes other multistep pathways to produce melanin [14].
A small amount of ultraviolet (UV) radiation is good for health, because it stimulates synthetic activity in the epidermis, but UV radiation can damage DNA causing mutation and promoting cancer development [28].Fortunately, the melanin in keratinocytes protects the epidermis and dermis from sunlight harmful effects caused by excessive amounts of UV radiation [28].However, the increased production and accumulation of melanin in the skin can be the cause of a large number of skin diseases, such as acquired hyperpigmentation that includes melasma, postinflammatory melanoderma, and solar lentigo [14], whereas it was found that there is a variations in human pigmentation among different racial groups because of the differences in the production and deposition of melanin in the skin [29], which may indicate that different races may have different prognoses for such diseases.However, dermatological diseases and their complications have a high frequency in many countries worldwide.Therefore, many researches have been carried out to better understand skin diseases pathways and mechanisms including the biochemical reactions implicated in mammalian melanogenesis [30] and the implication of excessive melanine production in numerous skin and health diseases [31].
Dermatological diseases not only affect the physical health of the patients, but also may have a psychological effect because of their visible nature [32], and even if the accumulation of melanin just darkens the skin without causing any disease, it may cause an appearance problem for persons not preferring dark skin.
Many melanogenesis inhibitors, which are used as cosmetic additives, are of limited effectiveness, difficult to formulate, or even cause reactions or undesirable effects after longtime usage [14].Thus, recently, there was more concentration towards the implication of natural products in cosmetics, which are usually characterised by their long-standing safety record [33][34][35].NG was one of the Chinese herbal medicines evaluated in order to find safer and more effective inhibitors of melanogenesis as treatments for diseases, or even as skinwhitening agents.
The effect of NG seeds on melanogenesis was investigated, and its methanol extract showed a dose-dependent inhibitory effect on the melanin synthesis of melanoma B16F10 cells [14].It has been shown that the methanol extract of this plant can inhibit melanogenesis significantly in a dose-dependent manner, and the MTT assay confirmed that the melanin inhibition activity is not caused by cytotoxicity, and methanol extract did not show any toxicity up to 100 g/mL [14].In order to identify the compounds involved in the melanin inhibiting effect of the methanol extract, solvent-solvent partition, silica gel open column chromatography, and GC-MS analysis were operated, and it was found that oleic acid methyl ester inhibited melanin synthesis in a dose-dependent manner without toxicity.Because oleic acid methyl ester is   the main active component in NG seeds, it is expected that the melanin inhibition activity is due to this compound [14].Another compound that was isolated in NG seeds and presenting an ability to inhibit melanin synthesis by inhibiting the tyrosinase enzyme activity in B16F10 melanoma cells is DOP [14].Since the methanol extract exhibited some toxicity towards the B16F10 melanoma cells, additional investigations on DOP relation with melanogenesis and its toxicity (MTT assay) have been conducted [14].They result in finding that this compound had a potent inhibitory effect on the melanogenesis of B16F10 melanoma cells (IC 50 = 24 M).Moreover, the compound also showed the same action mode as the crude extract of NG seeds [14].Although DOP has this inhibition effect of melanogenesis, it was classified as an endocrine disrupter recently, and its use is prohibited [36,37].
Usually, the presence of DOP in plants results from environmental pollution and many vegetables, such as Chinese tomato fruit that may contain it, and yet the cumulative content is less than 0.01 g/kg fresh weight [38].Therefore, the higher DOP content in NG seeds may indicate that NG plant possesses a stronger ability to accumulate DOP [14].It is worth noting that DOP may influence the results as a contaminant of solvents or plants [14].Moreover, further investigations are suggested to find out other compounds with depigmenting properties, isolated from NG seeds.[39].It is also the third leading cause of cancer death because of its very poor prognosis [40].Although numerous researches are focused on cancer treatment and many drugs are available as anticancer treatments, there is still a serious problem of chemotherapy, that is, multidrug resistance, which may cause the failure of cancer treatment by chemotherapy, in addition to the huge number of anticancer drugs side effects due to the lack of selectivity of anticancer drugs active compounds [2].

Anticancer Properties. Hepatocellular carcinoma is the most common cancer in many countries
These two problems are a great challenge for scientists to find out new drugs which are more selective and having as low toxicity as possible [41].Two oleanane triterpene saponins: --L-ara-binopyranosyl]-hederagenin), were isolated from NG seeds in this regard [2].Previous studies have reported that these two compounds display cytotoxicity in J 82, T 24, Colon 26 and 3LL cancer cells, with IC 50 values in the range of 1.1-16.5 M [42].In addition, kalopanaxsaponin A significantly reduced the LL/2 tumor weight of mice at an intraperitoneal dose of 5-10 mg and apparently prolonged the life span of mice bearing Colon 26 and 3LL Lewis lung carcinomas at 15 mg peritoneal dose [42].Kalopanaxsaponins A and I isolated from NG seeds cytotoxicity, drug resistance, and their selectivity were evaluated against two liver cell lines cell lines: HepG2, drug resistant HepG2 (R-HepG2) and primary cultured normal mouse hepatocytes [2].These two compounds showed significant cytotoxic activities towards the two cell lines and primary cultured normal mouse hepatocytes in a concentration-dependent manner [2].Their cytotoxicities on R-HepG2 were as effective as on HepG2, but the IC 50 values towards normal mouse hepatocytes were very low; consequently, these two compounds are effective against the drug resistant HepG2 cell line, but had no selective cytotoxicity towards cancer cells [2].Antitumor properties have also been studied for alpha-hederin.Although it is a commun constituent of NG and NS seeds, in this paper we emphasise the pharmacological activities investigated on NG seeds [43].
There are two different mechanisms of cell death, necrosis and apoptosis, which are different in morphology, mechanism, and incidence [44].Almost all the cytotoxic antitumor drugs induce apoptosis (programmed cell death), which is an important mechanism to investigate the possible anticancer activity of a compound [2].The inhibition mechanism for kalopanaxsaponins I and A was investigated through morphological observations and cell cycle analysis, and the findings showed that these two compounds can inhibit hepatoma proliferation by inducing apoptosis, and they may be considered as potential therapeutic agents for the treatment of parental and drug resistant hepatoma [2].

Anti-Inflammatory
Properties.Chronic inflammation is caused by various factors including infections (bacteria, viruses, or parasites), chemical irritants, and nondigestible particles.Since the inflammatory mediators can cause neoplasia, through inducing proneoplastic mutations, adaptive responses, resistance to apoptosis, or environmental changes [45], the development of anti-inflammatory therapy might be an efficient way to fight against cancer.The effect of NG seeds total saponins on inflammatory mediators and ERK/MAPK (extracellular-signal-regulated kinases/mitogen-activated protein kinases) pathway in stimulated macrophages was investigated.In order to study the anti-inflammatory mechanism, interferon-gamma (IFN-) plus LPS stimulated RAW 264.7 macrophages were used as inflammatory experimental model.The investigation of NG seeds total saponins anti-inflammatory effect was conducted through several assays [15].The production of the inflammation molecular mediator nitric oxide (NO), the total antioxidant capacity, mRNA expression, and protein expression examination were performed through Griess diazotization reaction, ferric reducing ability of plasma (FRAP) assay, reverse transcription polymerase chain reaction (RT-PCR), and Western blot, respectively, and the findings were that the saponins inhibited NO production in a dosedependent manner and suppressed the gene and protein expression of inducible NO synthase (iNOS).This inhibition of NO over production and iNOS expression was possible through ERK/MAPK pathway by noticeable inhibition of the phosphorylation of ERK (pERK) [15].Furthermore, NG seeds total saponins increased peroxisome proliferatorsactivated receptor PPAR- gene and protein expression, hence the increasing of anti-inflammatory mediators expression, whereas the total saponins contributed to the opposite effect on mRNA of inflammatory mediators, such as COX-2, IL-1, and IL-6, thus the increasing of the anti-inflammatory effect [15].

Antithrombosis, Antiplatelet Aggregation, and Ability to
Reduce Serum Triglycerides.Among the few studies in this regard, we illustrate two animal experiment examples that have been reported in the literature.To investigate these properties, homemade NG seeds oil emulsion was intragastrically administered to rats by 0.25 g/kg and 0.5 g/kg, with aspirin 0.9 g/kg as positive control drug, for nine days.Then, aortic blood was drew from each rat's abdomen, and platelet rich plasma (PRP) and poor platelet plasma (PPP) were prepared through centrifugation.Adenosine diphosphate (ADP) and collagen were used as inducers, and platelet aggregation was determined automatically with a turbidimetric method.Both of the two doses of NG seeds oil inhibited the induced rat platelet aggregation significantly [46].Another work was also conducted to study this effect, and rats were divided into five groups: two groups were the control and the model group, respectively, and for the other three groups, rats were administered aspirin intragastrically by 0.3 g/kg, NG seed oil by 0.5 g/kg, and 1.0 g/kg, respectively.The rats were administered the drugs for nine days successively, but they were administered cholesterol two days before.After the last administration of the drug, the rat's abdomen aortic blood was drew for blood clots experiment.It was found that NG seeds oil can significantly inhibit in vitro thrombosis of rats.The content of triglycerides and cholesterol in blood samples was also determined, and it was found that NG seeds oil can reduce the production of triglycerides, but no obvious effect on reducing cholesterol content has been noticed.Since the linoleic acid accounts for 55.35% in NG seed oil, it was suggested that it is the active ingredient in NG seeds oil that has the antithrombosis and antiplatelet aggregation effects, in addition to the ability to reduce the serum triglycerides.These results converge with the pharmacological activities believed in folk medicine [16].

Conclusions and Future Perspectives
Numerous plants are used in folk medicine and are popular worldwide.However, further investigations and clinical studies are still required to find out the active compounds, activity mechanisms, and possible toxicity; for instance, although NG belongs to the same family (Ranunculaceae) and the same genus as NS, researches focusing on the former are still lacking, compared to the latter.Furthermore, these two species seeds contain a number of similar and common compounds; for example, the alkaloids nigellicine and nigellidine were isolated from the seeds of NS and NG.Nigeglanine, with a special indazole ring, is another alkaloid isolated from NG seeds, which presents a chemical structure similarity with nigellicine and nigellidine due to the common indazole ring.Thymoquinone has also been isolated from both NG and NS seeds, and interestingly, this molecule has been described in a tremendous number of papers as a major bioactive component of NS seeds oil that affords a miraculous power of healing.The anti-inflammatory and anticancer properties of thymoquinone isolated from NS have been proven in vitro and in vivo.Moreover, other properties have also been reported as a potential treatment for various disease models including diabetes, asthma, and encephalomyelitis.Furthermore, this molecule could act as a free radical, superoxide radical scavenger, and preservation agent of various antioxidant enzymes activity, such as catalase, glutathione peroxidase, and glutathione-S-transferase [47].Alpha-hederin is another common constituent of NG and NS seeds.Studies have concluded that it has both hemolytic and antifungal activities [48].It has also a P450 suppression potential, which is apparently one of the mice protective mechanisms against chemicals-induced hepatotoxicity [49].It has also been investigated for its antitumor activity [43].However, these activities need deeper investigations, hence, the importance of conducting more studies not only on NS, which is described as a miracle herb [50], but also on NG seeds extracts that contain these active compounds.Nigellamine A 1 , nigellamine A 2 , and nigellamine B 1 are also common constituents of these two species seeds [51].They were isolated recently from NG seeds and are already known as constituents of NS seeds.These common compounds and other common constituents may reflect the potential similarity of pharmacological healing properties, which might be very useful for regions where NG is more available than NS.
Among the pharmacological activities of NS seeds oil and thymoquinone that were found out through researches on NS seeds are their protective effects on lipid peroxidation process during cerebral ischemia-reperfusion injury in rat hippocampus [52], their antinociceptive effects through indirect activation of the supraspinal mu(1)-and kappaopioid receptor subtypes in mice [53], and thymoquinone possible anticonvulsant activity in the petit mal epilepsy (probably through an opioid receptor-mediated increase in GABAergic tone) [54].Furthermore, on the basis of the structural similarity between compounds isolated from these two plants seeds, the similarity of their range of target diseases believed in folk medicine, and the fact that many therapeutical actions believed in folk medicine for NS seeds have been proven experimentally, and we strongly suggest that further research on NG seeds would be highly promising.Researches on NS seeds provide useful orientations for researchers towards elucidating the action mechanisms and thus finding out scientific explanations of NG seeds against numerous diseases.Therefore, we encourage conducting experiments on NG seeds on the basis of those carried out on NS seeds.We further suggest the study of the pharmacological activities that are already applied in folk medicine.Indeed, among the numerous studies and pharmacological properties investigated in NS seeds, we mention some selected examples.In pulmonology, the investigations included the study of the effects of NS seeds extract on ameliorating lung tissue damage in rats after experimental pulmonary aspirations [55], the considerable ability of NS oil to reduce the severity of lung damage due to hyperoxia [56], the ameliorative effect of NS oil on ovalbumin-induced bronchial asthma in mice [57], and NG potential ability of treating allergic rhinitis as an alternative when the effects of other antiallergic drugs need to be avoided [58].In microbiology, the protective effect of NS oil against murine cytomegalovirus infection [59] and the antimicrobial activity of diethyl ether extract of NS seeds against Gram-positive and Gram-negative bacteria have been studied [60].The effect of NS seeds oil against liver damage induced by Schistosoma mansoni infection in mice [61] was also investigated.NS ability to improve lipid profile and to prevent atherosclerosis in rabbits was also the subject for other studies [62].Other researches focused on the protective effects of NS against gentamicin-induced nephrotoxicity in rats [63].Experimental investigations have also been conducted to study NS aqueous and ethanolic extracts ability to stimulate milk production in rats [64].Furthermore, NS oil protective action against gastric secretion and ethanolinduced ulcer in rats [65], NS seeds volatile oil antioxytocic potential [66], the anti-inflammatory, the antipyretic activity of NS [67], and NS seeds effects on the central nervous system (CNS) and on analgesic activity [68] have also been studied.For all the mentioned examples, the results were highly encouraging and promising.The mentioned publications about NS in addition to other studies represent potential starting points to further the research on NG, which has an underestimated value despite the fact that NG and NS have been applied to treat almost the same diseases in folk medicine.
Further in vitro and in vivo investigations on NG seeds appear very promising, especially because of that the efficacy of this plants seeds on serious health problems, such as cancer, diabetes, thrombosis, and inflammation has been proven.NG seeds compounds could be a vital solution and even may suggest a whole food profile able to reduce NIDDM-induced pathogenesis directly, and indirectly through treating obesity problem.NG seeds represent a promising source of skinwhitening and antiobesity agents.These seeds constitute also a good source of unsaturated fatty acids, hence, the importance of their oil within healthy diet.
NG seeds might constitute a straight orientation on the right way to make a substantial amelioration in divers fields including nutrition, pharmacy, and aesthetics.However, further laboratory investigations and clinical studies remain highly required to investigate the safety and the toxicological profile of the isolated compounds, their in vivo efficacy, and their possible undesirable side effects.Moreover, further studies should be carried out to ensure the quality and the sustainability in regard of this amazing plant, which is highly promising to get a place among the scientifically toprated herbal medicines.In addition, we noticed that most of the published studies about NG have been conducted in China, especially west of China, and also in Korea.That is probably because of the historical and traditional use of NG in these two countries.However, collaborative research between divers countries' laboratories is recommended to generate reproducible results and to enlarge the research scale and enhance the chances of isolating novel compounds from this promising plant, and then scientifically prove the related activities that are believed and used in folk medicine.