Panax notoginseng: Pharmacological Aspects and Toxicological Issues

Current evidence suggests a beneficial role of herbal products in free radical-induced diseases. Panax notoginseng (Burk.) F. H. Chen has long occupied a leading position in traditional Chinese medicine because of the ergogenic, nootropic, and antistress activities, although these properties are also acknowledged in the Western world. The goal of this paper is to review the pharmacological and toxicological properties of P. notoginseng and discuss its potential therapeutic effect. A literature search was carried out on Pubmed, Scopus, and the Cochrane Central Register of Controlled Trials databases. The following search terms were used: “notoginseng”, “gut microbiota”, “immune system”, “inflammation”, “cardiovascular system”, “central nervous system”, “metabolism”, “cancer”, and “toxicology”. Only peer-reviewed articles written in English, with the full text available, have been included. Preclinical evidence has unraveled the P. notoginseng pharmacological effects in immune-inflammatory, cardiovascular, central nervous system, metabolic, and neoplastic diseases by acting on several molecular targets. However, few clinical studies have confirmed the therapeutic properties of P. notoginseng, mainly as an adjuvant in the conventional treatment of cardiovascular disorders. Further clinical studies, which both confirm the efficacy of P. notoginseng in free radical-related diseases and delve into its toxicological aspects, are mandatory to broaden its therapeutic potential.


Introduction
The first evidence of the medical use of crude herbs dates back to the mists of time when intelligent animals noticed that some food plants modified particular body functions.Human cultures institutionalized these observations so that the consumption of botanicals has often served important cultural traditions [1].Over the last 2-3 decades, however, the use of botanicals has overcome the limits of folk medicine, and several herbal products are currently used as add-on therapies for free radical-related diseases, such as neurodegenerative, immune-inflammatory and cardiovascular disorders, diabetes mellitus, and cancer [2][3][4][5][6].In this regard, herb-derived compounds, e.g., curcumin (the main ingredient of Curcuma longa Lynn.), ferulic acid (very abundant in Chinese herbs, including Angelica sinensis or dang gui, Cimicifuga foetida, and Ligusticum chuanxiong), ginkgolides (the active constituents of Ginkgo biloba), and ginsenosides (bioactive compounds abundant in ginseng species) have been extensively studied for their ability to scavenge reactive oxygen species (ROS) and enhance the cell stress response [7][8][9][10].
Ginseng is among the most popular botanical medications in both Europe and the United States of America (USA) because of its antioxidant, ergogenic, nootropic, and antistress activities [8,11,12].Botanical preparations of ginseng may result from several species of Panax, the most commonly used being Panax ginseng (P.ginseng or Korean ginseng) and Panax quinquefolius (P.quinquefolius or American ginseng) [8,13,14].Panax notoginseng (Burk.)F. H. Chen (P.notoginseng, also known as Chinese ginseng or Sanqi), grown in Yunnan and Guangxi, has long held first place in the whole patent medicines market in China, and the market volume of the single species has exceeded CYE 10 billion [15].Globally speaking, according to The Trade Vision LLC (Cheyenne, WY, USA), the top three supplier countries for P. notoginseng are China, Canada, and the USA, whereas the top three importing countries are the Philippines, the USA, and Vietnam [16].Lastly, the Global Notoginseng Root Extract Market report foresees a linear increase in the P. notoginseng world market over the 2024-2031 period, mainly as a pharmaceutical, cosmetic, and health supplement [17].
Available in many pharmaceutical forms, P. notoginseng has been used for the treatment of immune-inflammatory, cardiovascular, central nervous system (CNS), metabolic, and neoplastic diseases [15].However, some studies report significant interactions with commonly used drugs (e.g., anticoagulants and antiplatelet agents), thus raising concerns on drug-herb interactions [15].The aim of this paper is to review the pharmacological properties of P. notoginseng, focusing on pharmacognosy, pharmacokinetics, pharmacodynamics, and clinical studies demonstrating its efficacy in important diseases.Data on the toxicology of P. notoginseng will also be provided and discussed.
A literature search was carried out on the Pubmed, Scopus, and the Cochrane Central Register of Controlled Trials databases.The following search terms were used: "notoginseng", "gut microbiota", "immune system", "inflammation", "cardiovascular system", "central nervous system", "metabolism", "cancer", and "toxicology".Only peer-reviewed articles written in English, with the full text available, have been included.

Pharmacognosy of P. notoginseng
More than 200 bioactive compounds have been identified in P. notoginseng, including saponins, flavonoids, cyclopeptides, sterols, polyacetylenes, volatile oils, amino acids, and polysaccharides [15].Although these compounds are also common to both P. ginseng and P. quinquefolius, saponins, volatile oils (e.g., andrographolide), and polyacetylenes (e.g., panaxynol and panaxydol) are more abundant in P. notoginseng than in the other species [18].Saponins, mainly dammarane triterpenes, are the principal bioactive compounds and include both 20(S)-protopanaxatriol (PPT)-derived ginsenosides and 20(S)-protopanaxadiol (PPD)-derived ginsenosides [8,15,19] (Figure 1).PPD-derived ginsenosides R1, Rb1, and Rd, together with PPT-derived ginsenosides Rg1 and Re, account for more than 90% of the total saponins in P. notoginseng, but they are not specific because they can be found in other species of the Panax genus [20][21][22].However, among these saponins, ginsenosides Rb1 (30-36%), Rg1 (20-40%), and R1 (7-10%) are the most abundant and are currently considered as the standard compounds to evaluate the quality of P. notoginseng preparations [18,20,23].Despite this differential composition, the total saponin content in P. notoginseng is much higher than in P. ginseng (~15% versus 4%, respectively), thus suggesting that, at the same dose, there is a greater concentration of bioactive compounds in the former than in the latter species [18].P. notoginseng roots and rhizomes often undergo specific preservation processes, the most common being steaming.Notably, steaming results in the formation of novel ginsenosides, such as Rh4, Rk3, and Rg3, and this enrichment may be responsible for the specific pharmacological properties of steamed P. notoginseng [24,25].Indeed, according to the current literature, steamed P. notoginseng is beneficial in patients with hematopoietic and immune diseases, whereas raw P. notoginseng counteracts swelling, eases pain, and improves lipid metabolism [24,26].Interestingly, the ginsenoside composition in P. notoginseng may also vary depending on geographical distribution and climate factors.As shown by Liu et al., high annual precipitation and elevated humidity increase the content of ginsenosides R1, Rg1, and Rb1 [27].
Twelve male and twelve female healthy volunteers were treated with a single oral dose of P. notoginseng (90-270 mL of a water extract containing mainly ginsenosides Rg1, Rb1, Rd, R1, Re, Rh1, in decreasing concentrations) [32].At the highest dose, the most abundant saponin detected in the plasma was Rb1, which reached peak plasma concentrations (Cmax) of 21-35 nM within 8-12 h and was excreted with a half-life (T1/2) of 33-57 h [32].Importantly, compound K, absent in the ingested Sanqi extract, appeared in the plasma with a Tmax (the time necessary to reach Cmax) of 11-17 h and exhibited much greater bioavailability than the parent Rb1 [area under the curve0-t (AUC0-t) 4.75 µM•h and 0.75 µM•h, respectively] [32].The relatively higher bioavailability of compound K in Chinese women than in men may be explained by the prevalence of the Firmicutes and Actinobacter phyla, which include Eubacteria and Bifidobacteria, the genera involved in ginsenoside Rb1 deglycosylation and compound K formation [33].For a quantitative description of the main pharmacokinetic parameters, as well as gender-related differences, see Table 1.
Clinical studies investigating the pharmacokinetic profile of parenteral P. notoginseng are also available.In one of these studies, 12 healthy male volunteers were treated with P. notoginseng [as Xueshuantong, a formulation containing ginsenosides Rb1, Rd, Rg1, Re, and R1, obtained from Guangxi Wuzhou Pharmaceutical group (Wuzhou, Guangxi Zhuang Autonomous Region, China)] at the single dose of 150 mg either intramuscular (i.m.) or as 1.5 h intravenous (i.v.) infusion [34].Unlike oral administration, parenteral P. notoginseng did not undergo first-pass metabolism in the gut, and unchanged ginsenosides were detected in the plasma, the most abundant being Rb1 [34].This latter exhibited a mean Cmax of 6.1 µM and 9 µM with Tmax of 5 h and 1.5 h for i.m. and i.v.administration, respectively [34].Ginsenoside Rb1 underwent slow hepatobiliary excretion, and only 32-38% was excreted unchanged into the urine with T1/2 of 46-57 h [34].Similar results were described by Pintusophon et al., who treated 36 healthy volunteers (24 men and 12 women) with 250-500 mg Xueshuantong 2.5 h i.v.infusion [35].Once again, Rb1 was the most abundant ginsenoside detected in plasma with mean Cmax of 16-20 µM and 26-29 µM in subjects treated with 250 mg and 500 mg Xueshuantong, respectively [35].About 40% of Rb1 was excreted in the urines with T1/2 of 40-43 h [35].For a quantitative description of the main pharmacokinetic parameters, as well as gender-related differences, see
Twelve male and twelve female healthy volunteers were treated with a single oral dose of P. notoginseng (90-270 mL of a water extract containing mainly ginsenosides Rg1, Rb1, Rd, R1, Re, Rh1, in decreasing concentrations) [32].At the highest dose, the most abundant saponin detected in the plasma was Rb1, which reached peak plasma concentrations (C max ) of 21-35 nM within 8-12 h and was excreted with a half-life (T 1/2 ) of 33-57 h [32].Importantly, compound K, absent in the ingested Sanqi extract, appeared in the plasma with a T max (the time necessary to reach C max ) of 11-17 h and exhibited much greater bioavailability than the parent Rb1 [area under the curve 0-t (AUC 0-t ) 4.75 µM•h and 0.75 µM•h, respectively] [32].The relatively higher bioavailability of compound K in Chinese women than in men may be explained by the prevalence of the Firmicutes and Actinobacter phyla, which include Eubacteria and Bifidobacteria, the genera involved in ginsenoside Rb1 deglycosylation and compound K formation [33].For a quantitative description of the main pharmacokinetic parameters, as well as gender-related differences, see Table 1.
Clinical studies investigating the pharmacokinetic profile of parenteral P. notoginseng are also available.In one of these studies, 12 healthy male volunteers were treated with P. notoginseng [as Xueshuantong, a formulation containing ginsenosides Rb1, Rd, Rg1, Re, and R1, obtained from Guangxi Wuzhou Pharmaceutical group (Wuzhou, Guangxi Zhuang Autonomous Region, China)] at the single dose of 150 mg either intramuscular (i.m.) or as 1.5 h intravenous (i.v.) infusion [34].Unlike oral administration, parenteral P. notoginseng did not undergo first-pass metabolism in the gut, and unchanged ginsenosides were detected in the plasma, the most abundant being Rb1 [34].This latter exhibited a mean C max of 6.1 µM and 9 µM with T max of 5 h and 1.5 h for i.m. and i.v.administration, respectively [34].Ginsenoside Rb1 underwent slow hepatobiliary excretion, and only 32-38% was excreted unchanged into the urine with T 1/2 of 46-57 h [34].Similar results were described by Pintusophon et al., who treated 36 healthy volunteers (24 men and 12 women) with 250-500 mg Xueshuantong 2.5 h i.v.infusion [35].Once again, Rb1 was the most abundant ginsenoside detected in plasma with mean C max of 16-20 µM and 26-29 µM in subjects treated with 250 mg and 500 mg Xueshuantong, respectively [35].About 40% of Rb1 was excreted in the urines with T 1/2 of 40-43 h [35].For a quantitative description of the main pharmacokinetic parameters, as well as gender-related differences, see Table 1.Unfortunately, no clinical studies describing the pharmacokinetics of steamed P. notoginseng are currently available.Ginsenosides are metabolized in the liver, mainly through the 3A4 isoform of cytochrome P-450 (CYP), and excreted mainly through the feces, although the contribution of the kidney has been described (see above) [8,36].

Pharmacodynamics of P. notoginseng
Despite the extensive literature on the pharmacological effects of P. notoginseng, many studies provide only limited information because they report neither the exact chemical composition (e.g., ginsenoside type, amount) nor the degree of purification of the extract used.According to this premise, the current section focuses on the main preclinical studies describing the pleiotropic effects of P. notoginseng.

Immune System
The modulation of the immune response is one of the most beneficial effects of P. notoginseng.An ethanol extract from the P. notoginseng root, containing high levels of ginsenosides Rb1 and Rg1 (35% and 34% of the whole extract, respectively) at the doses 5-50 µg/mL, inhibited the lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in the murine dendritic cell line DC2.4 [37].Interestingly, ginsenoside Rg1 (50 µg/mL) inhibited the LPS-stimulated cytokine production by DC2.4 cells more effectively than Rb1 [37].
An ethanol extract of P. notoginseng, rich in ginsenosides R1, Rg1, Re, Rb1, and Rd (total content 76.20%), at the concentration of 200 µg/mL, decreased oxidative stress markers [nitric oxide (NO) and ROS] and enhanced the cell stress response (total glutathione and xanthine oxidase) in RD4/2 cells infected with porcine circovirus 2 (PCV2) [38].A similar decrease in ROS levels and enhancement of the cell stress response was detected in spleen lymphocytes of mice treated with 200 mg/kg intraperitoneal (i.p.) P. notoginseng extract 7 days after PCV2 infection [38].
P. notoginseng extract, in the range of 16-256 µg/mL, dose-dependently inhibited the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in monkey Marc-145 cells [39].Furthermore, at the dose of 10 mg/kg per os, this extract improved the immune response in piglets immunized with the anti-PRRSV JXA1-R vaccine, as observed by a marked decrease in PPRSV blood and tissue loads [39].An ex vivo arm of this study confirmed that the P. notoginseng extract upregulated interferon (IFN)-α and IFN-β production in piglet peripheral blood mononuclear cells isolated before the challenge and exposed to PPRSV [39].
Andrographolide was used as an adjuvant to potentiate the immunostimulating activity of influenza vaccine [40].As reported by Zhao et al., andrographolide (800 µg) boosted the immune response (e.g., splenocyte proliferation, bone marrow dendritic cell maturity, and both Th1 and Th2 cytokine secretion) in female Balb/c mice treated with the quadrivalent influenza vaccine [40].
An ethanol extract of steamed P. notoginseng (30-90 µM) inhibited migration and enhanced neutrophil apoptosis in a zebrafish tail-fin amputation model [24].In particular, Rk3, Rh4, and Rg3 ginsenosides-formed following the steaming procedure, as mentioned above-were the major compounds involved in the neutropenic response [24].
In a streptozotocin-induced diabetic rat model, a P. notoginseng extract (containing ginsenosides R1, Rg1, Re, Rb1, and Rd) at the doses of 40, 80, 160 mg/kg per os for 2 months, counteracted the increased serum levels of TNF-α, IL-6, and IL-1β and inhibited the overexpression of phosphorylated IκKβ, IκB, and p65, in the ocular tissue [42].Interestingly, ginsenosides Rg1 and Rb1 were detected in the ocular tissue at higher levels than the other saponins [42].In an in vitro arm of this study, ginsenosides R1, Rg1, Re, Rb1, and Rd (25 µM) suppressed the inflammatory responses by blunting NF-κB activation and signaling pathways in MIO-M1 cells [42].

Cardiovascular System
In a preclinical model of blood deficiency syndrome (obtained by injecting 0.07 g/kg cyclophosphamide for 3 days and then 0.02 g/kg acetylphenylhydrazine on the fourth day), mice were treated with steamed P. notoginseng powder (0.45-1.8 mg/kg per os for 12 days) [44].Steamed P. notoginseng supplementation increased erythropoietin and its receptor, thrombopoietin, granulocyte-macrophage colony-stimulating factor, and GATA-1 levels in mouse bone marrow, thus reversing anemia as shown by the normalization of leukocyte, erythrocyte, and platelet counts [44].Furthermore, steamed P. notoginseng favored the cell cycle reaction and activation of immune cells through the JAK-STAT pathway in mouse spleen, which could promote hematopoiesis [44].
Together with the blood-tonifying effect, steamed P. notoginseng has significantly more potent antiplatelet and anticoagulant effects than the raw extract.In a collagen-induced platelet aggregation model, steamed P. notoginseng extract (3 mg/mL) inhibited platelet aggregation of 55-89% in rabbit blood samples with greater efficacy than a raw extract [45].The antiplatelet effect increased with steaming duration and, after 9 h, was comparable to that produced by 200 µM acetylsalicylic acid (ASA) [45].The efficacy of P. notoginseng in inhibiting platelet aggregation is higher than that of P. ginseng and P. quinquefolius [45].Steamed P. notoginseng extract (3.3-10 mg/mL) increased prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time more than the raw extract at each of the tested concentrations [45].The antiplatelet and anticoagulant activity of steamed P. notoginseng extract, described in reconstituted systems, was confirmed in preclinical models.In Sprague-Dawley rats, a steamed P. notoginseng extract (500 mg/kg per os) inhibited platelet aggregation and increased bleeding time to a greater extent than the raw extract; interestingly, the percentage of platelet inhibition was not significantly different from the treatment with 25 mg/kg ASA [45].The mechanisms involved in steamed P. notoginseng antiplatelet and anticoagulant effects are diverse and related to the ginsenoside composition (Table 2).

Rk3
Inactivation of the integrin αIIb/β 3 and reduction of fibrinogen binding.Inhibition of MAPK and PI3K/Akt phosphorylation leads to reduced production of TXA 2 . [46]
In a preclinical model of atherosclerosis, such as apolipoprotein E knockout (ApoE-KO) mice fed with high-fat diet, P. notoginseng freeze-drying powder (containing ginsenosides R1, Rb1, Rg1, Re, and Rd, total concentration 85.3%), at the dose of 60 mg/kg i.p. for 8 weeks, reduced plaque area and lipid deposition in the atheroschlerotic lesions in the aorta [50,51].In addition, P. notoginseng reduced total cholesterol, low-density-lipoprotein cholesterol, and triglyceride blood levels in the treated animals, as well as the inflammatory response, as demonstrated by the inhibition of MMP-9, TIMP-1, and IL-1β [50,51].In this animal model, the effect of P. notoginseng on atherosclerosis can be comparable to that of 20 mg/kg simvastatin [50].An equivalent reduction in lipid deposition and inflammatory response was obtained with oral P. notoginseng powder [52].
In rats with middle cerebral artery occlusion (MCAO), a well-known animal model of ischemic stroke, a P. notoginseng extract (containing ginsenosides R1, Rg1, Re, Rb1, and Rd) in the dose range 40-80 mg/kg intragastric, reduced lipid peroxidation and potentiated the antioxidant activity in the brain tissue [54].In addition, P. notoginseng-treated rat brains exhibited marked upregulation of pro-survival pathways, including those related to either insulin growth factor 1 or phosphorylated Akt or phosphorylated mammalian target of rapamycin (mTOR) [54].Furthermore, in the same animal model, a not better characterized methanol extract of P. notoginseng, at the dose of 50 mg/kg i.p. 2 h after MCAO, reduced brain infarct volume and counteracted inflammation, as shown by a marked reduction of iNOS, cyclooxygenase (COX)-2, and NF-κB [55].

Central Nervous System
Rats treated with P. notoginseng powder (2.5 g/kg/day) 3 days before and 3 days after traumatic brain injury (free-fall method) exhibited a significant improvement in tissue injury and neural function at 72 h paralleled by a marked increase in mTOR and phosphorylated mTOR [56].This neuroprotective effect of P. notoginseng has been associated with a mTOR-dependent down-regulation of autophagy, as demonstrated by the reduction of sequestosome 1, beclin 1, and microtubule-associated protein 1 light chain 3 [56].In the same experimental model, P. notoginseng reduced both the severity of hemorrhage and brain inflammation, as observed by the reduced levels of PT and APTT, together with the inhibition of brain NF-κB [57].Saponins from the leaves of P. notoginseng (2.5-5-10 µg/mL), characterized by an increased content of ginsenoside Rc and Rb3, with a much lower content of ginsenosides Rb1, Rb2, and Rd, reduced 500 µM H 2 O 2 -induced cell death in primary cultures of rat cortical astrocytes [58].The antioxidant effects of P. notoginseng in astrocytes were associated with the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates the expression of several genes involved in the adaptive response to xenobiotics, including herbal products [58][59][60].In particular, P. notoginseng activated the heat shock response, as demonstrated by the enhanced expression of heme oxygenase-1, a promising target for neuroprotective agents [58,61,62].P. notoginseng-related neuroprotection was confirmed in the human neuroblastoma cell line SH-SY5Y exposed to oxygen and glucose deprivation/reoxygenation damage [58].The neuroprotective effects of P. notoginseng should not only be ascribed to saponins because polyacetylene compounds also play an important role.As shown by Wang et al., panaxynol (8 µM) promoted neurite outgrowth in NGF-differentiated PC12D cells with a dual mechanism involving both protein kinase A and mitogen-activated protein kinase (MAPK) activation [63].
Beneficial effects of P. notoginseng have also been reported in preclinical models of neurodegenerative disorders.Through the inhibition of mTOR signaling and autophagy activation, P. notoginseng saponins (R1, Rg1, and Rb1), at the concentration of 0.1-1 µg/mL, exerted neuroprotective effects in PC12 cells exposed to 20 µM β-amyloid (Aβ), an in vitro model of Alzheimer's disease (AD) [64].Ginsenoside R1 (5-25 mg/kg/day by gavage for 3 months), the third most abundant saponin in P. notoginseng, increased choline acetyltransferase expression and improved cognitive function in the APP/PS1 double-transgenic mouse model of AD [65].In this model, the mechanisms underlying the nootropic effect of ginsenoside R1 involve the upregulation of the insulin-degrading enzyme and the resulting inhibition of Aβ accumulation, both mediated by peroxisome proliferator-activated receptor-γ activation [65].Ginsenoside Rg1 (10 mg/kg/day i.p. for 3 months) reduced brain Aβ due to a marked inhibition of γ-secretase activity and preserved spatial learning and memory in transgenic AD mice overexpressing amyloid precursor protein (Tg mAPP) [66].In these animals, ginsenoside Rg1 enhanced the protein kinase A/cAMP response element-binding protein pathway activation, thus favoring synaptic function [66].

Metabolism
In a mouse model of type 2 diabetes mellitus, an ethanolic extract of P. notoginseng (rich in ginsenosides R1, Rb1, Re, Rg1, and Rd, 97.57%) at the dose of 20 mg/kg/day per os for 4 weeks, improved endothelium-dependent relaxations and alleviated endoplasmic reticulum stress and oxidative stress in aortas [70].P. notoginseng was also effective in improving glucose sensitivity and normalizing blood pressure in diabetic mice [70].The signaling pathways involved in these vasoprotective effects were those related to AMPK and endothelial NOS [70].In rats treated with alloxan (40 mg/kg i.v.), a preclinical model of type 1 diabetes mellitus, P. notoginseng saponins (no characterization was provided), at the dose of 10-200 mg/kg/day intragastric for 3 months reduced glycemia and lipid levels and restored kidney function [71].Furthermore, P. notoginseng treatment also restored the expression of both the bone morphogenetic protein-7 and plasminogen activator inhibitor-1, whose levels were reduced and increased, respectively, in the fibrotic kidney [71].In rat mesangial cells, P. notoginseng saponins (100 µg/mL) confirmed the anti-fibrotic effect by suppressing the transcription of transforming growth factor-β1 and monocyte chemoattractant protein-1, reversing the increased acetylation of NF-κB p65 by high glucose and increasing sirtuin-1 and superoxide dismutase (SOD) expression [71].
In a rodent model of obesity, such as mice fed on a high-fat diet (60% fat, 20% protein, 20% carbohydrate) for 2 weeks, P. notoginseng saponins (R1, Rg1, and Rb1, 83.84%), at the doses of 400 or 800 mg/kg/day per os for 7 weeks, modified the gut microbiota by increasing the abundance of Akkermansia muciniphila and Parabacteroides distasonis [72].Because of this reshaping, and due to the activation of the leptin-AMPK/STAT3 signaling pathway, an increase occurred in brown adipose tissue thermogenesis and beige adipocyte reconstruction [72].Lastly, reduced host adiposity and promoted energy expenditure were observed in these animals [72].4.6.Cancer P. notoginseng saponins Rb1, Rg1, Rd, R1, and Rh1, in the dose range 50-400 µg/mL, reduced the growth of Lewis-lung carcinoma (LLC) cell line, and mitigated the development of tumors in LLC cell-inoculated mice, by down-regulating a wide array of genes involved in malignant transformation, including Braf1, Cdk6, Notch3, Met, Col1a1, Hgf, Raf1, and Scd1 [73].
An ethanol extract of P. notoginseng, rich in ginsenoside R1 and Rg2, in the dose range 1-5 mg/mL, arrested the cell cycle at the G2/M phase in both prostate cancer LNCaP and 22Rv1 cell lines [74].In addition, P. notoginseng-treated PCa cells released less tumor-promoting IL-4 than controls [74].
Panaxynol and panaxydol (2 and 5 µM) have been shown to inhibit the proliferation of HL60 cells, a human promyeloblast cell line, through the activation of caspase-3 and the following cleavage of poly(ADP-ribose) polymerase [76].
With regard to colorectal cancer (CRC), P. notoginseng whole extract and ginsenoside R1 have been shown to inhibit migration, invasion, and adhesion of the human cell line HCT-116 [77,78].The anti-metastatic properties of ginsenoside R1 (75-300 µM) rely on the inhibitory effect on adhesion molecules such as MMP-9, integrin-1, E-selectin, and intercellular adhesion molecule-1 [77,78].P. notoginseng exerts adjuvant effects in CRC, as demonstrated by the evidence that n-butanol extract rich in ginsenosides Rb1, Rb2, Rb3, and Rc at the dose of 0.25 mg/mL enhances the antiproliferative effect of 5-fluorouracil on HCT-116 cancer cells and may decrease the dosage of the antiblastic drug [79].
Similar anti-metastatic properties of P. notoginseng saponins have been confirmed in an in vivo model of breast cancer.As shown by Wang et al., a mixture of ginsenosides Rb1, Rg1, Rd, R1, and Rh1 (50-400 µg/mL) induces apoptosis and cell cycle arrest of highlymetastatic mouse breast cancer 4T1 cells [80].In this cell line, P. notoginseng increases the expression of proteins that suppress tumor metastasis, including breast cancer metastasis suppressor 1, metastasis suppressor 1, and tissue inhibitor of metalloproteinase 2 [80].Comparable results have been shown in Balb/c mice injected with 4T1 cells, treated with P. notoginseng saponins (150 mg/kg i.p.), and sacrificed 3 weeks later [80].

Clinical Studies
Sanchitongshu is a Chinese medicine extracted from P. notoginseng and considerably rich in PPT-derived ginsenosides (~80%), 60% of which is Rg1 and the remainder Re (6%) and R1 (11%) [81].In a multi-center, double-blind, randomized clinical trial (RCT), 140 patients with ischemic stroke in anterior cerebral circulation were allocated to receive either ASA (50 mg daily) and 200 mg Sanchitongshu capsules (100 mg saponins and 100 mg inactive starch, Pharmaceutical Factory of Chengdu Hoist Inc. Ltd., Chengdu, China) three times daily, or ASA as above and placebo capsules for 4 weeks [81].Patients treated with the combination of ASA and Sanchitongshu achieved a significant improvement in both neurological deficits and activities of daily living compared with patients receiving ASA and placebo [81].Neither severe adverse effects nor deaths were reported during the RCT [81].Only eight patients in the Sanchitongshu arm and six in the placebo arm reported nausea, gastric discomfort, and increased frequency of evacuation lasting 2-7 days [81].
Xuesaitong, another Chinese medicine formula from P. notoginseng containing saponins Rg1 (35,3%), Rb1 (32.3%),R1 (9.80%), Rd (4.9%), Re (4%), and the remainder being other ginsenosides, has proven effective in ischemic stroke, cerebral infarction, and unstable angina pectoris [82,83].In a recent multi-center, double-blind RCT, 2966 patients with ischemic stroke were assigned to receive either Xuesaitong soft capsules (120 mg twice daily, manufactured by China Resources Kunming Shenghuo Pharmaceutical Co., Ltd., Kunming, China) or placebo for 3 months [84].Patients treated with P. notoginseng exhibited a significant improvement in neurological deficits and better functional independence than those receiving placebo (89.3% vs. 82.4%,respectively) [84].With regard to the safety profile, serious adverse events arose in 1.0% of patients receiving P. notoginseng and 1.1% in the control group [84].Xuesaitong has also shown great effectiveness in patients with cerebral infarction.A recent meta-analysis of 7 RCTs involving 827 patients concluded that Xuesaitong, as an add-on to conventional treatments (antihypertensive, hypoglycemic, hypolipidemic, antithrombotic, etc.), improved both clinical outcomes, such as the neurological deficit score and number of cured case, and blood rheology indicators, including whole blood viscosity, fibrinogen, and hematocrit, in patients with acute cerebral infarction [82].In the analyzed studies, no significant adverse reactions were reported.Likewise, a meta-analysis of 17 RCTs involving 2315 patients revealed that Xuesaitong combined with conventional medicines [ASA, β-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, statins, calcium-channel blockers, nitrates, etc.] significantly reduced mortality in patients with unstable angina with respect to those receiving only conventional treatment [85].In addition, Xuesaitong significantly reduced the duration of angina attacks and the dosage of nitroglycerin and improved both the electrocardiographic abnormalities and plasma lipid profile compared to conventional treatment [85].No adverse events related to Xuesaitong were reported in this study [85].In a single center, assessor-blinded RCT, 89 patients with stable coronary heart disease (SCHD) and endoscopy-confirmed chronic gastritis were assigned 100 mg of ASA daily or 60 mg of Xuesaitong capsules [Kunming Shenghuo Pharmacy Co., Ltd.(Kunming, China)] twice daily in addition to 100 mg of ASA for 8 weeks [86].Compared with ASA alone, Xuesaitong + ASA suppressed platelet COX-1 activity and decreased TXA 2 production [86].Furthermore, patients in the Xuesaitong arm exhibited a marked relief in dyspepsia-related symptoms, probably by increasing both gastrin and motilin release [86].No significant adverse effects were reported, with the exception of one patient in the Xuesaitong group, who experienced mild and transient constipation [86].

Interactions and Precautions
As mentioned in Section 3, P. notoginseng saponins are mainly metabolized through CYP3A4 [36], the isoform responsible for the metabolism of most drugs, and, therefore, several studies were carried out to explore the potential interaction between ginsenosides and this isozyme.Most available data derived from ex vivo studies show that P. notoginseng does not have any significant effect on the metabolism of conventional drugs metabolized through CYP3A4 [87,88].Similarly, negligible interactions have been reported in terms of induction/inhibition of CYP2C9, CYP2C19, CYP2B6, and CYP2E1 by P. notoginseng [87,89].Only limited information is available on the modulation of either phase II drug-metabolizing enzymes or transporters by P. notoginseng.Ginsenoside Rg3 inhibited UDP-glucuronosyltransferase isoforms 1A7, 1A8, 2B7, and 2B15 in a reconstituted system (K i 22.6, 7.9, 1.9, and 2.0 µM, respectively), whereas saponins Rh2, Rg3, and R1 inhibited P-glycoprotein (P-gp), thus reversing resistance to many drugs [87,90].In particular, ginsenoside Rh2 (5-50 mg/kg intragastric or 10 µM) increased the oral absorption of digoxin in rats and increased the bioavailability of ritonavir in Caco-2 and MDCK-MDR1 cells [87,91].The last ginsenoside, through either the inhibition of P-gp and NorA efflux pumps or the stabilization of the gyrase-fluoroquinolone complex, fosters the antibacterial effects of ciprofloxacin and levofloxacin towards susceptible bacteria, such as S. aureus [92][93][94].Lastly, a P. notoginseng suspension [containing ginsenosides R1 (6.9%), Rg1 (28.0%),Rb1 (29.7%),Re (3.8%), and Rd (7.3%)], at the dose of 30.25 mg/kg per os, increased the systemic bioavailability of ASA (20.83 mg/kg per os) in Sprague-Dawley rats [95].The mechanism for such an interaction relies on the ability of saponins to increase ASA transport across the cell membrane [95].
Although not yet proven in humans, this mechanism may increase the risk of bleeding in subjects treated with low-dose ASA who, at the same time, take P. notoginseng as a dietary supplement.
Although these results support the idea that P. notoginseng could enhance both the pharmacological or toxic effects of conventional drugs, these lines of evidence mainly derive from preclinical studies.The lack of significant data collected through clinical studies does not allow us to draw any conclusion on the potential herb-drug interaction of P. notoginseng.

Toxicology
The acute toxicity of raw and decocted extracts of P. notoginseng has been studied in zebrafish larvae.Both the raw and decocted P. notoginseng extracts dose-dependently killed larval zebrafish, with an estimated lethal dose (LD) 50 of 73.8 µg/mL and 151 µg/mL, respectively [100].Significant abnormalities in body weight, body length, and number of vertebrae were detected in larvae after 21 days of treatment with the decocted extract [100].However, the developmental toxicity of the decocted P. notoginseng extract seems to be related to neither the main compounds ginsenoside R1 nor the non-protein amino acid L-dencichine [100].Taking into consideration the lack of studies carried out in rodents, the main species for the study of xenobiotic acute toxicity, and the limited evidence provided by the study described above, it is not possible to draw any relevant conclusion on P. notoginseng acute toxicity.
With regard to sub-chronic toxicity, P. notoginseng powder (0.75-2.37-7.5 g/kg/day) was administered by gastric gavage for 90 days to 40 male Sprague-Dawley rats [101].Neither physical nor behavioral abnormalities were observed in treated animals [101].Compared to control rats, no significant differences were reported in body weight, total and differential white blood cell counts, total and albumin serum protein levels, transaminases, natural killer cell activity, lymphocyte transformation, spleen and thymus weight [101].
As far as the reproductive and genetic toxicity of P. notoginseng is concerned, unfortunately, no specific studies are available.The only evidence found in the literature is a study by Zhang et al., who evaluated the effects of P. notoginseng on platinum-based reproductive and genetic toxicity [102].The Authors reported that a P. notoginseng preparation containing ginsenosides Rg1, Rb1, and R1, administered at the doses 0.19-0.58g/kg/day for 14 days by gavage, counteracted cisplatinum-induced reduction in body weight and testis and epididymis weight in male Kunming mice [102].In addition, P. notoginseng increased sperm count/viability and decreased abnormal sperm morphology in cisplatin-treated animals [102].Lastly, P. notoginseng reduced both the tail moment and micronucleus formation in peripheral blood cells and bone marrow cells, respectively, in cisplatin-treated mice [102].

Conclusions and Future Directions
As reported in the pharmacodynamic studies summarized above, P. notoginseng exerts beneficial effects by acting at several levels.The beneficial effects described on immuneinflammatory, cardiovascular, neurodegenerative and metabolic diseases, as well as in cancer, make P. notoginseng an important herbal product.Unfortunately, despite the abundance of preclinical data, few clinical trials supported the therapeutic role of P. notoginseng, mainly as an add-on therapy for the treatment of cardiovascular diseases.The good news from these studies is that, at the controlled doses, P. notoginseng has nearly no toxic effects.Some concerns arise when considering the folk use of P. notoginseng as a dietary supplement.In 1979, Siegel reported a "ginseng abuse syndrome" characterized by CNS hyperactivity, skin eruptions, and morning diarrhea in patients taking high-dose ginseng (up to 15 g/day) [103].These lines of evidence, together with the limited data about the toxicological profile of P. notoginseng, downsize its translational potential.Indeed, in 2020, the European Food Safety Agency (EFSA) considered a botanical extract derived from P. notoginseng and Astragalus membranaceous safe at the dose of 0.5 mg/kg/day in adults, excluding pregnant women [104].Although promising, this EFSA report is based on data related to a mixed extract of both P. notoginseng and Astragalus membranaceous, without any possibility of identifying the profile of single products [104].Sub-chronic and chronic studies carried out in male and female rodents and beagle dogs would help assess the effects of long-term exposure to P. notoginseng.In addition, these studies would allow us to calculate the lowest observed adverse effect level (LOAEL) and no observed adverse effect level (NOAEL), parameters necessary for deciding the dose to use in controlled human studies.Only thanks to this approach can ginseng arise from the niche of a dietary supplement or traditional Chinese medicine to gain drug status as suggested by preclinical studies.

Table 1 .
Quantitative description of the main pharmacokinetic parameters of ginsenoside Rb1 and compound K in humans treated with different preparations of P. notoginseng.For further information, see Section 3.
Data are expressed as mean ± standard deviation; a 270 mL; b 150 mg; c 1.5 h intravenous infusion; d 2.5 h intravenous infusion; AUC 0-t , area under the curve 0-t ; C max , peak plasma concentration; N.C., not calculated.PO, per os; T 1/2 , half-life; T max , time to reach the C max.

Table 2 .
Main mechanisms through which steamed P. notoginseng ginsenosides exert antiplatelet and anticoagulant effects.For further information, see Section 4.3.