We examined all phytochemical papers published in English by using PubMed, Google Scholar, Elsevier and Science Direct databases. For reviewing functional properties of Piper plants, in vitro/in vivo studies from the year 2013 to 2018 have been collected. The part which summarizes clinical trials has focused on anxiolytic properties of specific kava extracts between 1991 and 2013 and has only covered studies with monotherapy.
7.1. Antiproliferative/Anti-Cancer Properties
According to recent world health statistics published by WHO, cancer caused 9.0 million deaths in 2016 [306
]. It is expected that this number will increase in coming years unless successful treatment and/or preventive strategies can be developed [307
]. Mankind has traditionally used plants to self-treat health problems throughout history; nowadays researchers focus on understanding the action mechanism of plant-based chemicals and identifying effective sources for new drugs [308
]. Naturally occurring plant-based chemicals serve alternative approaches regarding chemotherapy and chemoprevention with relatively fewer side effects. In the present section, in vitro and in vivo studies involving evaluation of the anti-proliferative, anticancer and chemopreventive effects of both extracts and bioactive constituents from Piper
plants were reviewed.
Dried samples of P. longum
were extracted with several solvents including hexane, benzene, acetone, ethyl acetate, ethyl alcohol, chloroform, and water [309
]. The anticancer activities of the various extracts were evaluated in several different human carcinoma cell lines (A549 lung, THP-1 leukemia, DU-145 prostate, IGR-OVI-1 ovary, and MCF-7 breast cancers). The cytotoxic activities of the extracts (100 μg/mL) quantified by sulforhodamine B assay on those cell lines. All extracts produced growth inhibition in THP-1 (76–90%); while hexane and benzene extracts showed cytotoxic effect (>80%) on the growth of all cell lines. It was noted that standard anticancer drugs exhibited 51–67% cytotoxicity in comparison with control groups in the study. Acetone, benzene and hexane extracts inhibited cell cycle 43, 41 and 63%, respectively in A549 cell line and resulted in increased sub-G1 DNA fraction population. The effectiveness of extracts was also studied on Wistar rats having AlCl3
-induced hepatotoxicity. Accordingly, aqueous, chloroform and ethyl alcohol extracts showed protective effect on liver 65, 71 and 64%, respectively against peroxidative damage. In another study, ethanol extract of fruits of P. longum
were examined by using in vitro and in vivo models to clarify its efficacy and safety [310
]. Interestingly, the results indicated that treatment by extract selectively induced cell death in carcinoma cells including HCT116 colon, BxPC-3 pancreas and T cell leukemia; however extract was not effective on normal colon epithelial cells. Findings were supported by Annexin V Binding Assay confirming that ethanolic extract induced cell death by caspase-independent apoptosis. As animal models, immunocompromised mice were studied, and administered with ethanolic extract at a dose of 50 mg/kg/day routinely for 6 weeks. In treatment group, the growth of colon cancer tumors was suppressed without any toxic effect.
In vitro and in vivo studies demonstrated that P. umbellatum
species can be a promising source for anticancer agents. In the study of Iwamoto et al. [311
], the extraction of milled fresh leaves of P. umbellatum
was prepared by dichloromethane. Total growth inhibition was achieved in several different human carcinoma cell lines (U251, MCF-7, NCI-H460, UACC-62, PC-3, 786-0, NCI-ADR/RES and OVCAR-3) with relatively small effective doses (6.8 and 14.9 µg/mL). As the concentration of total growth inhibition was determined as 144.6 µg/mL for nontumor cell line, cytotoxic effect of dichloromethane extract seem to be selective for tumor cells. In the same study, Balb/C mice with Ehrlich solid tumor were treated with 100, 200, and 400 mg/kg of the extract by oral route. They found that the sizes of the tumors were reduced 38.7 and 52.2% in 200 and 400 mg/kg extract treatment groups, respectively, without toxicity. Similar effects were also shown by another study in which crude extract of P. tuberculatum
treated as a test material [10
]. The cytotoxic activity IC50
of the crude extract was found to be 10 μg/mL in SF-295 cells and 4.3 μg/mL in the HCT-8 cell line. Test material was also examined in Balb/C nude mice (100–200 mg/kg/day); as a result, a hollow fiber assay showed that crude extract inhibited cell proliferation in those SF-295 and HCT-8 cell lines by 24.6–54.8%, respectively, without any sign of toxicity.
In the study of de Souza Grinevicius et al. [312
], the ethanolic extract of P. nigrum
fruits was evaluated for its antitumor activity in Balb/C mice model of Ehrlich carcinoma and breast (MCF-7) and colon cancer (HT-29) cell lines. The dytotoxicity of the extract was determined in carcinoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and effective concentrations (EC50
) of 27.1 ± 2.0 µg/mL in MCF-7 and 80.5 ± 6.6 µg/mL in HT-29 were found. In vivo studies exhibited that extract treatment elicited 60% decrease of tumor growth and 76% increase of survival time together with stimulation of apoptosis which collectively resulted in inhibition of Ehrlich carcinoma in mice. Furthermore, Bax and p53 protein expression levels were increased, whereas Bcl-xL and cyclin A expressions were inhibited with the treatment of extract; so, cell cycle arrest at G1/S and increased rate of apoptosis were observed. The following year, a more effective extraction method—Supercritical Fluid Extraction (SFE)—was introduced; briefly a high-pressure unit was used to obtain extract from the P. nigrum
fruits, specifically Bragantina cultivar [313
]. SFE produced an extraction product, namely SFE200, having a minimum ratio of monoterpenes/ sesquiterpenes and a relatively higher concentration of piperine. SFE200 showed higher cytotoxic effects on MCF-7 cells than conventional extracts. In vivo studies validated the effect of this special extract for which Ehrlich tumor-bearing mice were selected as model organism. Treatment by SFE200 at a dose of 10 mg/kg/day presented more significant tumor growth inhibition and increased the time for survival comparing with conventional extract treatment group. It was also noticed that SFE200 decreased cell number founding at S phase, probably due its apoptotic effect by arresting G2/M phase of cell cycle.
In contrast to the above study where the piperine content was increased, the biological effects of piperine-free P. nigrum
(PFPE) extract were investigated in the study of [314
]. Several cancer cell lines and two normal cell lines were subjected to different doses of PFPE to determine its cytotoxicity and selectivitiy index. Among cell lines, PFPE showed higher cytotoxic effect on MCF-7 breast carcinoma cells (IC50
: 7.45 µg/mL) together with better selectivity. Flow cytometry analysis demonstrated that PFPE induced apoptosis on MCF-7 carcinoma cells in a dose-dependent manner. The expressions of apoptosis-associated proteins were also examined by western blot, and treatment by PFPE with IC50
concentration upregulated p53 and cyt C and downregulated topoisomerase II. Similar results were also obtained for in vivo studies. When rats with mammary tumorigenesis were administered with 100 mg/kg PFPE, tumor grew much smaller as compared to non-treated control group. The same research group published one more article in the following year that clarifies the action mechanisms of PFPE on breast cancer [315
-methylurea-stimulated mammary carcinoma rat models were administred with different concentrations of PFPE (100, 200 and 400 mg/kg). The most significant tumor suppression activity was obtained in 400 mg/kg treated groups and suppression scores were 2.18-fold and 1.75-fold for 100 and 200 mg/kg treatment groups, respectively. Moreover, PFPE decreased the expression level of vascular endothelial growth factor (VEGF), E-cad and c-Myc proteins as compared to control groups, while protein level of p53 was significantly increased. Western-blot analyzes over breast cancer cell line MCF-7 exhibited similar results for those proteins but expressions of proteins were not changed significantly in PFPE-treated ZR-75-1 cell line. In conclusion, PFPE was found to be a promising extract for the suppression of uncontrolled proliferation of cancer cells through increasing the expression level of p53 and by decreasing the level of c-Myc.
Not only Piper
extracts but also major bioactive components of Piper
plants, mainly piperine, piperlongumine, and flavokawain B (FKB), have been investigated as potential anticancer agents in several studies. Piperine is a kind of pungent alkaloid found in P. nigrum
and P. longum
]. Besides its anticancer effectw, it has been reported as an anti-inflammatory, neuroprotective and cardiovascular protective agent [317
]. Furthermore, its anti-angiogenic characteristic was shown for the very first time in 2013 [321
]. According to this study, piperine inhibited the proliferation of HUVEC cells via inhibiting G1/S transition, cell migration and tubule formation without toxic effect. As compared to control groups, 100 μM piperine treatment inhibited the activation of PKB through phosphorylation from the residues of Ser 473 and Thr 308 in HUVEC cells, whereas it had no effect on protein expression of TRPV1. In ex vivo test design, inhibition of tubule development was achieved in the treatment group of rat aorta angiogenesis model.
In the study of Yaffe et al. [322
], piperine treatment on HRT-18 human rectal adenocarcinoma cells inhibited metabolic activity in a dose-dependent manner. Flow cytometric analysis represented that piperine also induced apoptosis. However, in this study it was found that apoptosis induced by piperine was mediated by the increase in reactive oxygen species (ROS) production; particularly, hydroxyl radicals. Treatment of cells with N
-acetylcysteine, a well-known antioxidant, suppressed apoptosis in piperine-treated cells.
Piperine exhibited its antitumor effects by inhibiting HER2
mRNA expression l in HER2
-overexpressing breast carcinoma cell lines [323
]. In piperine-treated cells, apoptotic cell death increased by caspase-3 activation and PARP cleavage. Piperine also reduced cell migration by interfering with several signaling pathways including Akt, ERK1/2 and p38 mitogen-activated protein kinase (MAPK). Piperine demonstrated similar effects on vascular smooth muscle cells (VSMCs) found within blood vessels by inducing arrest at cell cycle and suppressing the activation of the MAPK through phosphorylation [324
]. It reduced the BB and platelet-derived growth factor (PDGF)-induced uncontrolled cell proliferation by changing the expression of p27Kip1
, cell cycle proteins including cyclin E, cyclin D and PCNA.
Piperine was found to be an anticancer agent against osteosarcoma cells including HOS and U2OS cell lines [325
]. It showed growth inhibitory action in both osteosarcoma cell lines but relatively weaker inhibitory effect in normal hFOB cells. By piperine treatment, the cell population found in G2 phase was enhanced while the cell population in G1 phase was reduced, however, no changes in S phase population were observed. Therefore, piperine exerted its effect by arresting cell cycle at G2/M phase. Additionally, piperine represented its inhibitory properties on cell migration and invasion by increasing the expression of TIMP-1/-2 and by down-regulation of MMP-2/-9.
In another study, it was reported that piperine has potent antitumor activity against triple-negative breast cancer (TNBC) [326
]. Both in vitro and in vivo analyses presented its selective inhibition effect on cell growth. Controlled cell death induced by piperine was shown through the mitochondrial pathway and through the suppression of Akt activation in breast cancer cells. In a recent study, piperine exhibited antitumor property against human ovarian cancer cell line (A2780) [327
]. Cell viability was reduced in piperine-treated cancer cells whereas no significant changes were seen in normal cell line. The proportions of phosphorylated forms of JNK and p38 MAPK protein to non-phosphorylated forms were increased by piperine treatment in dose-dependent manner suggesting that JNK and p38 MAPK mediate intrinsic apoptotic pathway in ovarian cancer cells.
, also known as kava-kava, is another Piper
species rich in three kinds of chalcones, namely flavokawain A, B, and C. Among these compounds, FKB has been reported as promising anti-inflammatory, antinociceptive, and as well as antitumorigenic agent toward several cancer cell lines [328
]. Antitumorigenic effects of FKB were evaluated in both 4T1 cell line and Balb/C mice [332
]. Cytotoxicity of FKB toward 4T1 cell lines was determined by MTT assay and cell growth was inhibited in a dose-dependent manner. Additionally, FKB interfered with cell cycle and raised the cell population in the SubG0/G1 phase as compared to control groups. Findings from in vivo study also showed the inhibition of tumor growth in 50 mg/kg FKB treated mice during 28 days. Interestingly, FKB treatment increased CD4/CD3 T-cell and CD8/CD4 T-cell populations significantly resulting in positive effect on immune system of test animals. Apart from that, FKB treatment reduced the cancer formation in another side of body including lung, liver, and spleen significantly while the number of colonies was higher in the non-treated group. Antiangiogenic effect of FKB has been investigated in 1.0, 2.5, and 5.0 μg/mL treated HUVEC cell lines [333
]. Tube-like structures were counted manually and accordingly branch formations were found to be 38.44 ± 3.95 and 25.44 ± 5.69 in 2.5 and 5.0 μg/mL treated groups, respectively. In the same study, zebrafish embryos were subjected to FKB as test model. Up to 5.0 μg/mL FKB treatment, there were no toxicity signals. However, 10 μg/mL inhibited the formation of subintestinal veins and showed toxic effect. Therefore, the best inhibition rate was observed in 2.5 μg/mL FKB-treated group. These reports for promising anticancer activities of FKB provided the inspiration for the synthesis of potentially more effective FKB derivatives. Accordingly, in the study of Abu Bakar et al. [334
], 23 FKB analogs were synthesized. The cytotoxic effects of these compounds were evaluated in two breast cancer cell lines (MCF-7 and MDA-MB-231). Five synthetic derivates showed notable cytotoxic activities in MCF-7 cell line (IC50
5.5–5.9 µg/mL). Piperlongumine, also known as piplartine, is another bioactive component, an amide alkoloid found in the fruits of Piper
species, particularly, in P. longum.
While it was isolated in 1961 for the first time, its pharmacological properties including neuroprotective, anti-atherosclerotic and antimicrobial, have been elucidated in the past decade [335
]. Although there were two in vitro studies showing its potent antiproliferative effects on both prostate and ovarian cancer cell lines in 2013 and 2014, the mechanism based detailed studies for its anticancer properties were published recently. The antiproliferative effects of PL at low micromolar concentrations on LNCaP and PC-3 human prostate cancer cells was found to be associated with reduction in the protein level of androgen receptor AR, which is key element of oncogenic precursor [337
]. PL also inhibited cell growth in human ovarian cancer cells with IC50
values in the ranges of 6 to 8 µM in three different cell lines by G2/M cell cycle arrest [338
]. In those cells, intracellular ROS levels were increased by PL treatment in dose dependent manner which consequently resulted in apoptosis.
Pro-apoptotic effect of PL treatment was also seen in an experimental design with human cholangiocarcinoma cell lines in concentration dependent manner [339
]. In this study it was shown that the activation of caspase-3, PARP, JNK-ERK as well as stimulation of ROS accumulation underlies the action mechanism of PL. Furthermore, PL treatment induced p21 expression at protein level resulting cell cycle arrest at G2/M phase in cholangiocarcinoma cell lines (KKU-055, KKU-100, KKU-139, KKU-213, and KKU-214). Similar anticancer potency was seen in 5 to 15 μM PL-treated pancreatic, lung, kidney, and breast cancer cell lines [340
]. Protein analysis showed that PL suppressed expression of Sp1, Sp3, Sp4, and Sp-regulated genes containing cyclin D1, survivin, cMyc, EGFR and hepatocyte growth factor receptor (cMet). Apoptosis was shown to be ROS-mediated and both were found to be attenuated after co-treatment with glutathione.
Recently Machado et al. [341
] reported that PL did not show a genotoxic effect on plasmid DNA and CT-DNA assessed by cleavage activity and circular dichroism assays. However, studies on HCT 116 cells exhibited ROS-mediated apoptotic activity of PL.
In the study of da Nóbrega et al. [342
], nineteen PL analogues have been synthesized by using the 3,4,5-trimethoxycinnamic acid-like starting material, and their cytotoxic potencies were screened in U87MG glioblastoma cell line. Among these test compounds, (E)
-benzhydryl 3-(3,4,5-trimethoxyphenyl) acrylate, which has two aromatic rings in the side-chain, presented the best inhibition effects on viability of U87MG cell line in a dose dependent manner by oxidative and apoptotic processes. In this study, the biosafety of this compound was also assessed by sister chromatid exchange and 8-hydroxy-20-deoxyguanosine assays in human peripheral blood cells. Results indicated that this synthesized compond would be promising an agent selectively cytotoxic and genotoxic to cancer cells as well as with strong bioavailability. Taken together, some extracts and active constituents of Piper
plants can be promising as antiproliferative and chemopreventive agents on which more in vivo studies as well as well-designed clinical trials are crucially needed.
7.2. Anti-Inflammatory Properties
Inflammation can be considered as a defense mechanism of living organisms to various stimuli involving pathogens, toxic compounds, and many environmental stress factors [343
]. Normally, this highly regulated and self-limiting phenomenon promotes the healing process. However, uncontrolled and pro-longed process, called as chronic inflammation, may cause sustained activation of immune cells resulted in high amount of cytokine release [344
]. This persistent response potentially leads to pathological disorders such as autoimmune diseases, diabetes, heart diseases, cancer and various neurodegenerative disorders. Understanding the basis of inflammation and characterization of novel anti-inflammatory agents may serve promising therapeutic approaches for the prevention and/or treatment of chronic inflammatory diseases.
Functional biological activities of Piper
plants against inflammation were shown by numerous in vivo and in vitro studies. In the study of Tasleem et al. [346
], ethanol and hexane extract of P. nigrum
and one of its bioactive compound, piperine, were shown to be effective against carrageenan induced-paw edema in Swiss albino mice. Piperine treatment inhibited edema growth at all doses (5, 10 and 15 mg/kg). While the hexane extract was effective at 5 and 10 mg/kg treatment groups, the best inhibition score was obtained at 10 mg/kg treated group for 60 min. The ethanol extract also exhibited good inhibition profile at 10 mg/kg treatment group as compared to control groups. However, all inhibition scores were less than the standard drug, diclofenac sodium.
To elucidate the effects of P. nigrum
ethanolic extract (PNE) on airway inflammation model, Balb/C mice were induced with ovalbumin (OVA) and administered orally with 200 mg/kg PNE [347
]. PNE decreased the size of inflammation related cells such as eosinophils, goblet, and mast cells in bronchoalveolar lavage fluid (BALF). Although OVA induced the cytokine productions, PNE regulated balance of T cells responses by decreasing IL-1β, IL-4, IL-17A, and TNF-α cytokine levels in both BALF and lung homogenate. Thus, PNE demonstrated significant potency on inhibition of inflammation.
Laksmitawati et al. [348
], investigated the anti-inflammatory activity of Piper crocatum
extracts prepared by maceration technique using 96% ethanol. To screen the toxic effect, murine RAW 264.7 macrophage cell line was treated with several doses of extract (0–500 μg/mL) and subjected to MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H
-tetrazolium) assay. In 150 μg/mL, and higher treatment concentrations, cell viability was found to be significantly affected as compared to control groups. TNF-α level was decreased significantly at 50 μg/mL treated cell group while each treatment groups (10, 50 and 75 ug/mL) showed decreased IL-1β levels as compared to control groups. Extract at 10 and 50 μg/mL concentrations exhibited significant reduction in IL-6 levels and the lowest NO level was obtained in the 50 μg/mL treatment group.
In the study of Reddy et al. [349
], the analgesic and anti-inflammatory activity of a hydroalcoholic extract (70% ethanol and 30% distilled water) of P. betle
leaves (HEPBL) were demonstrated. Wistar rats and Albino mice were administered different concentrations of HEPBL according to OECD guideline no. 425 to detect non-toxic treatment concentrations. Treatments at 100 mg/kg and 200 mg/kg exhibited significant analgesic properties in both animal groups. Interestingly, HEPBL decreased the pain by both central and peripheral mechanisms. According to literature nonsteroidal anti-inflammatory drugs inhibit the pain just peripherally, but narcotic analgesics reduce the pain both centrally and peripherally. HEPBL also showed anti-inflammatory effect against the carrageenan induced paw edema model and cotton pellet-induced granuloma model. Compared to control group, HEPBL significantly inhibited the paw edema growth in the 50, 100, 200 mg/kg treatment groups in a dose dependent manner. The most effective dose was shown to be 200 mg/kg with a 79.73% reduction score after 3 h. Similarly, reduction in the dry weights of granuloma was reported as 57.49% in a 200 mg/kg treated group as compared to control groups.
A standardized dichloromethane extract (SDE) of P. umbellatum
leaves exhibited anti-inflammatory activity on carrageenan-induced paw edema and peritonitis models [311
]. Balb/C mice were administered with carrageenan solution (2.5 mg/mL, 40 µL/animal) to induce inflammation, and after one hour 100, 200, and 400 mg/kg of SDE were treated. The paw volume was measured at certain intervals. SDE treatment demonstrated inhibitory effect on paw edema during first phase (24 h) without toxicity. Interestingly, increased leukocytes migration promoted second phase of inflammation. However, all concentrations of SDE inhibited inflammation at 48 h. Taken together, P. umbellatum
SDE might be promising anti-inflammatory agent.
Recently, Finato et al. [350
] investigated the anti-inflammatory effect of crude extracts of P. gaudichaudianum
, P. arboreum
, P. umbellata
, P. fuligineum
and, Peperomia obtusifolia
in LPS-induced peripheral blood mononuclear cells (PBMCs). Cytotoxic concentrations of extracts were determined by MTT assays and IC50
(mg/mL) values of 0.55, 2.19, 1.56, 0.24, and 2.12 were found for each extract, respectively. These specified doses were treated to cells with and without an inflammatory stimulus; after 24 h incubation, cell-free supernatants were subjected to cytokine analysis. All extract exhibited differential inhibitory effect on proinflammatory cytokines such as IL-1β, IL-6, IL-8, IL-10 as well as TGF-β1 and TNF-α.
In addition to Piper
extracts, bioactive components of Piper
plants were also evaluated in terms of their anti-inflammatory activities. In the study of Umar et al. [351
], piperine exhibited anti-inflammatory effects in collagen-induced arthritis model of Wistar rats that were administered with 100 mg/kg of piperine for 21 days. Effectiveness of piperine on production of inflammatory mediators (IL-1β, TNF-α, IL-10 and PGE2
) were assessed by ELISA and by Griess assay for measurement of NO. Piperine treatment reduced the NO production in piperine group as compared to control group. Moreover, administration of piperine inhibited the production of pro-inflammatory mediators, IL-1β, TNF-α and PGE2
, but increased the level of IL-10.
In the study of Ying et al. [352
], the effects of piperine at non-toxic doses (10, 50 or 100 μg/mL) were investigated in LPS induced RAW264.7 cells. As compared to LPS treatment, piperine reduced the production of PGE2
and NO significantly in dose-dependent manner. Additionally, protein expression levels of COX-2 and inducible NOS were suppressed significantly at 50 or 100 μg/mL, while their mRNA expression levels were decreased at each treatment group. Piperine exhibited the inhibitiory effect on both TNF-α production and its mRNA expression in dose dependent manner. Moreover, piperine treatment caused a significant decrease in the activation of NF-ĸB by suppressing IĸBa phosphorylation and a reduction in the nuclear translocation of p65 subunit.
Additionally piperlonguminine (PL), a bioactive molecule of P. longum
demonstrated protective effects against endothelial barrier disruption induced by LPS-stimulated-proinflammatory responses in cell and animal models [353
]. Barrier protective effects of PL (between 5–40 µM doses) have been investigated by measuring endothelial cell permeability, migration and monocyte adhesion assays as well as by measuring the activation of proinflammatory proteins in LPS-induced HUVEC cells and in mice. PL reduced the migration of monocyte cells to HUVECs, expression of CAMs (cell adhesion molecules) thus demonstrated a protective effect against LPS-stimulated disruption of endothelial barrier. It was also demonstrated that PL inhibited LPS-induced production of IL-6 and TNF-α through inhibiting the activation of NF-ĸB and ERK 1/2 by LPS.
Kava (P. methysticum)
extracts are commonly used as a preventive strategy for the treatment of various mental disorders such as anxiety and nervous tension. There are also experimental evidences substantiating anti-inflammatory effects of this species or its active components [354
]. In the study of Kwon et al. [356
], one of the active chalcone constituents of kava extracts called flavokawain A (FKA) has been investigated for its anti-tumor and anti-inflammatory activities in LPS-induced RAW 264.7 macrophage cell line. Accordingly, FKA pretreatment resulted in reduced production of LPS-induced NO and PGE2
levels compared to LPS-treated control. Besides, protein and mRNA expression levels of iNOS and COX-2 were also suppressed by FKA treatment in a dose-dependent manner. FKA also inhibited LPS-induced activation of NF-kB. Moreover, FKA downregulated LPS-induced pro-inflammatory cytokines, including IL-6, IL-1β and TNF-α at both protein and mRNA level.
Another kava component, kavain and its structural analog kava-241 obtained synthetically also investigated for their anti-inflammatory properties [357
]. In this study, RAW 264.7 cells were exposed to 100–300 μg/mL of kava and kava-241 after 0.1 μg/mL of LPS stimulation. While kava reduced LPS-induced TNF-α production by 75%, kava-241 resulted in a more prominent inhibition (85%) on the same parameter as compared to control group. Moreover, kava-241 showed less cytotoxic effect than kava. In vivo studies demonstrated that in the periodontitis model of DBA1/BO male mice, 40 mg/kg kava-241 administration resulted in reduction of epithelial downgrowth (72%) and alveolar bone loss (36%) as compared to untreated-control groups. In the study of Huck et al. [358
], kava-241 was found to be effective against Porphyromonas gingivalis
-induced joint inflammation in a murine arthritis model. In the mentioned study, animals were treated with 50 μg/100 µL of P. gingivalis
-LPS and 600 µL of kava-241 (40 mg/kg) for 17 days. Kava-241 treatment reduced the size of inflammatory cells and osteoctlasts in the side of inflammation. Moreover, Kava-241 inhibited the TNF-α production and TLRs protein expressions through the suppression of activation of ERK, MAPK, AKT and p38 proteins. Collectively, these studies support claims that Piper
plants are potential candidates for treatment of inflammation-based diseases.
7.3. Neuropharmacological Activities
Uncontrolled systemic inflammation may proceed into a persistent chronic state that may have neurological impacts in the development of many neurodegenerative disorders including PD and AD [359
]. Recent studies showed that therapeutic compounds acting on single targets (e.g., acetylcholinestaerase inhibitors) often show insufficient efficacy and undesirable toxic effects in the treatment of neuroinflammatory CNS disorders [360
]. Studies suggest that balanced modulation of several interconnected targets can be more efficient strategy than the single target modulation for treating complex neuroinflammatory disorders with multi-factorial nature [360
]. Plant species often serve good sources for the identification these kinds of multitarget agents. Extracts of Piper
plants and their active compounds especially piperine, piperlongumine and kavalactones have been extensively investigated for the prevention and the treatment of neurodegenerative diseases through in vitro [363
] and in vivo [365
] studies as well as well-designed clinical trials [367
]. In the last five years, the in vitro and in vivo studies for the effect of extracts on neurodegeneration models included P. nigrum
, P. betle
and P. sarmentosum
species. Evidences from in vitro experiments support promising effects of last two species against the manifestations of neurodegenerative diseases.
is an abundantly found Piper
species in certain regions of the world, particularly tropical areas. Ferreres et al. [363
], investigated the phenolic profiles and anti-cholinesterase activity of both aqueous and ethanol extracts of leaves. Regarding chemical composition, hydroxychavicol was identified as a major phytochemical in both extracts. Both extracts demonstrated potent inhibitory efficacy against acetyl- and butyrylcholinesterase enzymes. In this study, the effects of extracts on the viability of neuronal cells (SH-SY5Y) were also evaluated at the mitochondrial function (MTT reduction) and membrane integrity (LDH release) levels. Accordingly, ethanol extract between 7.8–1000 μg/mL concentrations did not result in an alteration in the membrane integrity or the function of mitochondria. However, the aqueous extract at 125 µg/mL reduced the cell viability about 20% and interfered with the mitochondrial function. For this extract, more significant reductions in cell viability were reported above 500 μg/mL concentrations which were shown to be cytotoxic to neuroblastoma cells (human). The results of this study suggest that P. betle
leaf extracts could be promising for the prevention/treatment of neurodegenerative disease.
(PS) is one of the edible, terrestrial species of Piper
plants, abundantly found around the Asian regions. Traditionally, it has been extensively used for the treatment of various CNS disorders such as anxiety, depression and memory dysfunctions. Previous studies reported that P. sarmentosum
has anti-depressant, anti-inflammatory, anti-oxidant and anti-acetylcholinesterase activities [364
]. In the recent study of Yeo et al. [364
], in vitro cytoprotective properties of different extracts from leaves of P. sarmentosum
against Aβ-induced microglia-mediated neurotoxicity were investigated. Inhibitory effects of four extracts ethyl acetate (LEA), hexane (LHXN), dichloromethane (LDCM) and methanol (LMEOH) on Aβ-induced production and mRNA expression of some pro-inflammatory factors in BV-2 microglial cells were assessed. Additionally, the protective effects of extracts on human neuroblastoma cells (SH-SY5Y) were also evaluated by using Aβ-induced conditioned media from microglia cells. The LEA and LMEOH extracts resulted in reduction in the secretion levels of Aβ-induced pro-inflammatory cytokines (IL-1β and TNF-α) by downregulating the mRNA expressions of pro-inflammatory cytokines in BV-2 cells. LEA and LMEOH pre-treated conditioned media from microglia cells elicited protection on human neuroblastoma cell line against Aβ-induced neurotoxicity through downregulation of phosphorylated tau proteins. The results of this in vitro study suggest that polar extracts of P. sarmentosum
leaves could be a promising complementary alternative in the treatment of AD.
Functional properties of P. sarmentosum
as antidepressant were exhibited in rodent animal model by means of hypothalamic-pituitary-adrenal axis regulation [373
]. In the study of Li et al. [373
] these properties of the P. sarmentosum
extract and its ethyl acetate fraction (PSY) were investigated by using several parameters including forced swimming test, open field test, and tail suspension test in mice. The results showed that treatment of mice with either P. sarmentosum
extracts at 100 and 200 mg/kg or PSY at 12.5–50 mg/kg doses resulted in potent antidepressant effects, which is similar to response obtained by 20 mg/kg conventional therapeutic drug fluoxetine. Moreover, PSY increased the level of BDNF protein as well as phosphorylation levels of CREB and ERK proteins in the hippocampus of rats suggesting that P. sarmentosum
can modulate the physiology of brain cells.
(black pepper) is among the most extensively studied species for its neuropharmacological activities. Anxiolytic, antidepressant, neuroprotective and antineuro-inflammatory effects of P. nigrum
extracts have been examined in multiple animal studies. The methanolic extract prepared from the seeds of P. nigrum
was investigated in management of AD stimulated by neuroinflammation in the rat model [374
]. In this study, aluminum chloride (AlCl3
) at 17 mg/kg b.w was used orally for one month to induce an AD model. Thereafter, animals in the group of AD were divided randomly into subgroups such as AD control; positive control (orally administered with a conventional drug-rivastigmine) and extract group (orally administerd with P. nigrum
extract). Postmortem brains of the animal were examined for several parameters such as levels of monocyte chemoattractant protein-1 (MCP-1), acetylcholine, C-reactive protein (CRP) and NF-κB. Administration of AD rats with P. nigrum
extract resulted in substantial improvements in the above-mentioned parameters which suggest that it may have potent anti-neuroinflammatory effects and may be promising in the treatment of AD.
In another study, the methanolic extract of P. nigrum
was analyzed for its memory-enhancing and antioxidant properties in AD models of rats which were experimentally induced by amyloid beta (1–42) [375
]. Rats were administered with extract at 50 and 100 mg/kg doses orally for 21 days. The memory-enhancing effects of the plant extract were studied by Y-maze and radial arm-maze tasks approaches. The antioxidant activities of the extract in the hippocampus were assessed by using superoxide dismutase-, catalase-, glutathione peroxidase-specific enzymatic assays and the total content of reduced glutathione (GSH), malondialdehyde, and protein carbonyl levels. Significant reduction in spontaneous alternations percentage within Y-maze task and increase of working memory and reference memory errors within radial arm-maze task were reported for AD group. However, treatment with the plant extract significantly ameliorated memory performance and showed remarkable antioxidant prospect.
Hritcu et al. [365
], also elucidated anxiolytic and antidepressant properties of the methanolic extract of P. nigrum
in a β-amyloid (1-42) rat model of AD. The mentioned effects of the extract were evaluated by numbers of open-arm entries, forced swimming tests and the time spent in open arms. In the Aβ (1-42)-treated AD models of rats, the number of open-arm entries together with percentage of the time spent in the open arms were significantly decreased which indicated that the Aβ (1-42)-treated rats experienced high levels of anxiety and they represented an efficient model to show the anxiolytic effects of extract. Accordingly, the administration of the methanolic extract increased the time spent in the open arms of Aβ (1-42)-treated rats significantly. The number of open-arm entries for Aβ (1-42)-and methanolic extract-treated rats increased as compared to the Aβ (1-42) treated group. The forced swimming test is one of the validated tools for predicting the antidepressant properties of drugs [376
]. Regarding this, the swimming time significantly increased in the Aβ (1-42)- and methanolic extract-treated group as compared to the Aβ (1-42)-treated group. Taken together, results demonstrated that treatment with the methanolic extract elicited a marked anxiolytic and antidepressant effects.
There are important specialized phytoactives found in Piper
species such as flavanones, chalcones, dihydrochalcones, and alkaloids as mentioned before. Piperine (1-piperoylpiperidine), a nitrogenous pungent alkaloid, is one of the major functionally active constituents responsible from neuropharmacological activities of P. nigrum.
The neuroprotective, anti-neuroinflammatory and anti-depressant effects of piperine have been elucidated in multiple animal studies. In 6-hydroxydopamie (6-OHDA)-induced PD model of Wistar rats, Shrivastava et al. [366
], reported that piperine treatment reduced neuronal cell apoptosis at a remarkable rate through inhibition of poly(ADP-ribose) polymerase activation and pro-apoptotic Bax levels as well as through the elevation of Bcl-2 levels. Furthermore, it was shown that piperine treatment reduced cytochrome-c release from mitochondria and diminished caspase-3 and caspase-9 activation induced by 6-OHDA. Treatment with piperine also caused a marked reduction of 6-OHDA-induced lipid peroxidation and stimulation of GSH levels in striatum brain region of rats. In this study, piperine also reduced the level of pro-inflammatory cytokines namely, TNF-α and IL-1β, in 6-OHDA-induced PD model of rats. Therefore, this study suggests that piperine has highly potent neuroprotective effect through its strong antioxidant and anti-inflammatory properties as wells as anti-apoptotic mechanism of action in 6-OHDA induced PD models.
Previous studies showed that piperine produces antidepressant-like effects through the inhibition of enzymatic activity of monoamine oxidase and increasing the levels of monoamine neurotransmitters in various mouse models of behavioral despair [324
]. Two studies from the same research group also reported that intraperitoneal administration of piperine to mice caused a significant reduction in immobility time, an important parameter of serotonergic system, assessed by forced swim and tail suspension tests [378
]. To clarify the molecular mechanism(s) underlying the antidepressant-like action of piperine Mao et al. [380
] examined the effect of piperine treatment on depressive-like behavior and brain-derived neurotrophic factor (BDNF) protein expression in the hippocampus and frontal cortex of mice exposed to chronic unpredictable mild stress (CUMS). 10 mg/kg chronic treatment of piperine significantly attenuated behavioral deficits in CUMS mice evaluated by forced swimming and sucrose preference tests. In mechanism-based studies, piperine treatment significantly increased the expression level of BDNF protein in the hippocampus and frontal cortex of both naïve and CUMS mice. The antidepressant-like effects of piperine in CUMS mice were significantly blocked by the injection of K252a, an inhibitor of BDNF receptor (TrkB), suggesting that ameliorative potential of piperine are mostly related with its capacity to increase the BDNF level. In a different study Mao et al. [380
], it was also reported that corticosterone-induced depression like behavior of model mice can be successfully suppressed by pretreating animals with piperine (at 5 and 10 mg/kg) for 21 days as assessed by diminished sucrose consumption which was found to be related with the elevated expression level of BDNF protein in the hippocampus. Regarding all these findings, it may be inferred that piperine exerts its antidepressant-like effect through modulating BDNF signaling.
Piperine treatment at 10 mg/kg also exerted a neuroprotective effects against a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) (MPTP)-induced mouse model of PD [381
]. Accordingly, piperine treatment ameliorated the MPTP-induced disruptive effects in motor coordination and in cognition-related functions. It also reversed MPTP-induced decreases in the number of tyrosine hydroxylase-positive cells in the brain region of substantia nigra. In addition, piperine diminished the number of activated microglia and reduced the IL-1β expression and oxidative stress. Piperine also demonstrated an anti-apoptotic mechanism of action by restoring the imbalance between Bcl-2 and Bax proteins. The results of this study may suggest that piperine can be a promising therapeutic treatment alternative for PD due to its potent neuroprotective activities on dopaminergic neurons by means of anti-apoptotic and anti-inflammatory mechanisms of action.
An imbalance between autophagy and apoptosis has been detected in PD patients. While mitochondrial dependent apoptosis increases [382
], the rate of autophagy decreases in the samples obtained from their brain [384
]. Agents to restore this imbalance of apoptosis and autophagy have been under investigation as treatment strategy for PD. Liu et al. [386
] reported that piperine elicits a cytoprotective activity in rotenone-induced SK-N-SH cells, primary rat cortical neurons and in mouse models by inducing phosphatase 2A (PP2A) and restoring the imbalance between autophagy and apoptosis. In SK-N-SH cells and primary neurons, piperine caused elevated cell viability and maintaining of mitochondrial functioning. Four week piperine administration (at two different doses; 25 and 50 mg/kg) ameliorated rotenone-induced disruption of motor functions and saved the loss of dopaminergic neurons in the substantia nigra region of PD mice models. In addition, it was reported that the rate of autophagy elevated by suppression of mammalian target of rapamycin complex 1(mTORC1) and activation of PP2A. A similar mechanism of action for piperine was also demonstrated in rat models. Wang et al. [387
] showed that apoptosis was diminished in the presence of piperine, however, autophagy was stimulated therefore abolishing neuronal injury in the rotenone-induced rat model of PD. Two important results represented by both rat and mice models of PD in above-mentioned studies suggest a novel understanding for the neuroprotective action mechanism of piperine in the treatment or prevention of neurodegenerative disorders.
Piperine also exhibited strong anti-neuroinflamatory effect on LPS induced BV2 microglia cell line [388
]. In this study, it was showed that piperine significantly inhibited LPS-induced TNF-α, IL-6, IL-1β, and PGE 2 production in BV2 cells through inhibition of NF-κB and activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Recently, it has been suggested that chronic neuro-inflammation may lead to pathological amyloid β (Aβ) and τ accumulations in late-onset AD [389
] which can be modelled in animals by intracerebroventricul-streptozotocin (ICV-STZ) treatment [390
]. Recently it has been shown that piperine has relatively selective effects on cognition in the ICV-STZ animal model and it causes improved hippocampal function prior to significant Aβ deposition [390
]. Accordingly, it was reported that the cognitive-enhancing effect induced by piperine at a relevant dose was simultaneous with hippocampal malonaldehyde decrement and the redox balance. In addition, histopathological outcomes were in accordance with the neuroprotective properties of piperine.
Taking all the abovementioned studies together, it can be concluded that piperine possesses profound effects on neurodegenerative disorders of the CNS such as AD and PD. However, the oral delivery of piperine to the brain is hampered due to many pharmaceutical challenges such as low water solubility, extensive first-pass metabolism and low absolute oral bioavailability [391
]. Recently, the use of brain-targeted nanosystems has been extensively investigated to improve delivery characteristics of challenging lipophilic active compounds. Studies have shown that piperine encapsulation can be achieved using different solid lipid nanoparticles and results have indicated that piperine-loaded nanoparticles remarkably reduce the severity of neurodegenerative diseases modelled by experimental animals [391
]. In a recent study by Etman et al. [393
], piperine-loaded oral microemulsion as a nanosystem increased piperine efficacy and enhanced its delivery to the brain resulting in better therapeutic outcome compared to the free drug in male Wistar rats exposed to ICV-colchicine injection to induce sporadic dementia of Alzheimer’s type. In another recent study by Anissian et al. [394
], piperine-loaded chitosan-sodium tripolyphosphate nanoparticles enhance the neuroprotection and ameliorate the astrocytes activation in chemical kindling model of epilepsy.
Piperlongumine (PL), an alkaloid amide, is the major active constituents of long pepper (P. longum
). Piperlongumine has previously shown to have anti-inflammatory and anticancer activities [395
]. Recent studies also showed that piperlongumine possess highly potent anti-neuroinflammatory functions. In vitro studies showed that piperlongumine significantly attenuated the production of proinflammatory mediators (NO and PGE2
) and some cytokines (TNF-α and IL-6) by suppressing NF-κB signaling pathway in LPS induced BV-2 microglias, primary astrocytes, RAW264.7 macrophages and Jurkat cells [13
]. Some important insights into the anti-neuroinflammatory and neuroprotective effects of piperlongumine have come from recent studies using animal models. Accordingly, piperlongumine inhibited LPS-induced memory impairment, Aβ accumulation and the activities of β- and γ-secretases in murine models [397
]. Gu et al. [398
], reported that the paralytic severity and neuropathology in mice model of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein 35–55 immunization were reduced in piperlongumine-treated group in comparison with the EAE model group.
There are also some in vitro and animal studies showing neuroprotective effects of piperlongumine and its analogs in case of neurodegeneration. In the study of Peng et al. [399
], two synthetic analogs of piperlongumine elicited low cytotoxicity and potent protection in hydrogen peroxide- and 6-hydroxydopamine-stimulated cell damage in the neuron-like PC12 cells via increasing the cellular levels of some antioxidant molecules. In rotenone-induced PD mouse models, Liu et al. [390
] showed that four week piperlongumine (2 and 4 mg/kg) administration attenuated motor deficits and prevented the loss of dopaminergic neurons in the substantia nigra. In the same study, it was also indicated that piperlongumine improved cell viability and enhanced mitochondrial function in primary neurons and SK-N-SH cells through inhibition of apoptosis and induction of autophagy. Therefore, we can note that similar to piperine, piperlongumine also exerts its neuroprotective effects on PD by setting the disrupted balance between two key parameters namely, apoptosis and autophagy.
There is also some evidence to suggest that piperlongumine can be an efficient strategy to improve cognitive functions. In recent studies, piperlongumine has been reported to increase cognitive function in a transgenic mouse model of AD as well as hippocampal activities and cognitive dysfunction of aged mice [400
]. In the hippocampus region of the aged mice, piperlongumine resulted in substantial elevation in the levels of calmodulin-dependent protein kinase II alpha (caMKIIα) and ERK1/2. Furthermore, following piperlongumine treatment in the dentate gyrus of the hippocampus the level of neurogenesis was significantly potentiated which was assessed by counting doublecortin-positive cells [401
]. In regard of these results, piperlongumine can be suggested as a promising bioactive compound in treatment and prevention of age-related cognitive impairment and hippocampal changes.
Long standing neuropharmacological activities of P. methysticum
including anxiolytic, sedative, muscle relaxant, mild anaesthetic and analgesic effects can be mostly attributable to kavalactones, particularly yangonin, kawain, dihydrokawain and methysticin, found in the lipid soluble fractions of the extracts [402
]. Several in vitro and in vivo studies have elucidated possible biological action mechanisms of kavalactones including blockade of voltage-gated sodium and calcium ion channels, enhanced ligand binding to γ-aminobutyric acid A (GABAA
) receptors, reduced neuronal reuptake of noradrenaline (norepinephrine) and dopamine, as well as weak inhibitory action on monoamine oxidase-B. Recently, Fragoulis et al. [403
] reported that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in transgenic mouse model of AD.
The medicinal use of kava extracts obtained from the root and rhizome parts as anxiolytic preparation has extended around the world since 1990 [404
]. However, due to issues related with hepatotoxicity, the use of kava was forbidden by the Federal Institute of Drugs and Medical Devices of Germany in 2002. In the same year, Food and Drug Administration of USA recommended a consumer warning but never banned the use of kava [405
]. Currently, in the USA kava extracts can be found in markets and on the internet. However in Germany, although the decision of court about the banning of kava use was found an inappropriate action in 2014, currently, it can be used only under the order of a referring practitioner [404
7.4. Clinical Studies
Clinical studies on Piper plants have been largely focused on the use of kava extracts for treating anxiety disorders due to its widespread and as well as restricted uses around different regions of the world. Kava extracts prepared from the root and rhizome part of the plant have strong clinical records which substantiate its efficacy as anxiolytic agent.
The earliest randomized, placebo-controlled, clinical trial was performed on 58 anxiety patients with non-psychotic origin [367
]. 100 mg kava extract (WS1490, a pharmaceutical extract) or placebo preparation was administered to patients daily three times during four week period. Drug receiving group demonstrated a significant reduction in overall score of anxiety symptomatology assessed by Hamilton-Anxiety-Scale (HAMA) as main target variable just after one week of treatment. The difference between drug and placebo group increased in the course of study. No adverse experiences were observed during the treatment period of kava extract.
A randomized, multicenter, placebo-controlled, double-blind clinical trial was carried out over a period of 25 weeks with 101 anxiety patients/non-psychotic origin [368
]. All patients administered with either 110 mg kava extract (WS1490) or placebo three times a day. Drug receiving group showed a significant superiority in overall score of anxiety symptomatology assessed by Hamilton-Anxiety-Scale over placebo starting from week 8. Evenly distributed adverse events were observed rarely in both groups.
Another randomized, double-blind, placebo-controlled clinical trial was conducted on thirteen patients with generalized anxiety disorder (GAD) [407
]. Patients were administered with kava 280 mg/day (standardized to 30% kavalactones) or placebo for 4 weeks. In this study two indication of vagal control which were defined as baroreflex control of heart rate (BRC) and respiratory sinus arrhythmia (RSA) were assessed. Accordingly, more patients showed significantly improved BRC following treatment with kava than with placebo. The magnitude of improvement in BRC was found to be significantly correlated with the degree of clinical improvement. In contrast, treatment with kava did not alter the magnitude of RSA, a measure of the heart rate changes occurring with respiration.
Mood disturbances, particularly anxiety and depression, are frequent in the premenopausal period [408
]. Although, they spontaneously vanish with time, their manifestation may have great impact on the quality of life in women [410
]. Hormone replacement therapy benzodiazepines, and antidepressants may ameliorate mood, but these pharmacological approaches sometimes may be associated with side effects and frequently be non-accepted by the women [409
]. Kava extract has been proposed to exert positive effects on mood, particularly, anxiety of premenopausal women. In a 3-months randomized, prospective open study, the effects of Kava extract at two different doses (100 mg and 200 mg/day) were investigated in 80 perimenopausal women [413
]. Several parameters such as anxiety assessed by state trait anxiety inventory scale, depression evaluated by Zung’s scale and climacteric symptoms shown by Greene’s scale were measured after first and third months. A placebo group was not included to the study and to compare the effects of Kava extract, a control group was used. As a result, in kava-treated groups, while anxiety and climacteric score declined at first and third months, depression was reduced at the third month. However, in the control group anxiety, depression and climacteric symptoms also showed decreasing tendency, so these findings were not significant.
In a double-blind and placebo-controlled trial, 50 anxiety patients with non-psychotic origin were treated by kava extract (WS 1490) 150 mg/daily for 4-week of period followed by 2-week observation phase to draw significant information on dosage range, safety, and efficacy of the preparation. For the primary efficacy variable assessed by HAMA score, kava treatment resulted in a significant reduction in anxiety as compared to placebo. For the secondary variables evaluated by HAMA subscales ‘somatic anxiety’ and ‘psychic anxiety’, a statistically significant advantage of the kava treatment over placebo was found to be detectable [414
]. At the beginning and end of the trial, the laboratory tests demonstrated no pathological changes in the generalized biochemical parameters such as blood counts, hepatic enzyme levels, total bilirubin, glucose, and some lipid profiles. Therefore, it was concluded that the special kava extract can be classified as a well-tolerated and safe preparation without any drug-induced adverse reactions or symptoms associated with withdrawal.
In another double-blind clinical trial which was also carried out to investigate safety and efficacy profile of the same pharmaceutical kava extract (WS1490), 61 patients with sleep disturbances associated with anxiety, tension and restlessness states of non-psychotic origin were treated with daily doses of 200 mg extract or placebo over a period of 4 weeks [415
]. ‘Quality of sleep’ and ‘Recuperative effect after sleep’, assessed by sleep questionnaire SF-B, demonstrated statistically significant group differences in favor of kava extract. Efficacy of kava was also indicated in the treatment of anxiety evaluated by HAMA scores. More prominent effects in terms of well-being based on self-rating and of global clinical assessment were also shown for kava extract. In clinical and laboratory parameters no differences were observed among groups. Besides, no drug-related adverse events or changes were seen. Safety and tolerability of kava were reported as good. Therefore, beyond the general anxiolytic effect of kava extract demonstrated in various controlled clinical trials this study evidenced that sleeping disorders related to non-psychotic anxiety may be treated by kava extract in an effective and safe ways.
An additional study to establish efficacy and safety of an aqueous extract of kava, a 3-week placebo-controlled double-blind, cross-over trial, specifically named as The Kava Anxiety Depression Spectrum Study (KADSS), was handled [416
]. Sixty adult patients with 1 month or more of elevated GAD were prescribed daily five kava tablets (each containing 3.2 g, standardized to 50 mg of kavalactones) or placebo. Kava conferred a decrease of 11.4 points as compared to placebo on HAMA score. In addition, significant decrease in Beck Anxiety Inventory and Montgomery—Asberg Depression Rating Scale scores showed that it may also have antidepressant effects. In terms of safety concerns, extract did not resulted in serious adverse reactions.
According to a WHO commission report (Organization 2007) evaluating the safety of kava products special water-based preparation should be preferred over extracts obtained by organic solvents. In addition, more studies are needed for the developments of standardized aqueous extracts which should be validated by well-designed controlled clinical trials.
In a placebo-controlled, double blind, randomized clinical study the aqueous extract of kava was evaluated for the efficacy outcomes on anxiety, as well as was assessed on a range of secondary outcomes including liver function tests, withdrawal or addiction and female’s sexual drive [417
]. Seventy five patients with GAD were administered with one tablet of kava twice a day (delivering 120 mg of kavalactones per day which could be increased to two tablets twice per day in non-response group-delivering 240 mg of kavalactones) for 6-weeks. The study contained a matched placebo group. Kava treatment resulted in significant reduction of anxiety as measured by HAMA score compared to placebo group [417
]. No significant adverse reactions were found in kava group. In terms of liver function tests, no significant differences were detected across groups. Besides, no variations were reported between groups in terms of withdrawal or addiction behavior. According to Arizona Sexual Experience Scale (ASEX), kava resulted in significant increase in female sexual drive as compared to placebo. In males, negative effects were not reported. Interestingly, it was found that there was a highly significant correlation between improved sexual function and performance (decrease in ASEX score) and anxiety reduction in the whole sample. As a part of this trial, they also examined GABA transporter polymorphisms as potential pharmacogenetic markers of kava response and found that specific polymorphisms appear to potentially modify anxiolytic response to kava [418
The same research group in 2015 extended the scope of the clinical trial and the number of participants [419
]. The secondary outcomes enriched by several parameters including genomic and neuroimaging techniques. A bi-center, 18 weeks, randomized, double blind, placebo-controlled phase III study were designed. 210 currently anxious participants with diagnosed GAD who were non-medicated were administered with aqueous extract of kava (standardized to deliver 240 mg of kavalactones per day) or placebo. The trial has been completed by 2018. However, the results of this study have not been released yet.
Collectively, several recent well-designed clinical trials confirmed the potency and safety of kava extracts as anxiolytic preparations. However, although kava extracts can be a valuable and rational therapeutic options for patients suffering from anxiety and related disorders, physicians must be aware of the range of issues including different quality of kava extracts, patient’s liver function and simultaneous use of other medications before prescribing [418