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Evaluation of molecular chaperons Hsp72 and neuropeptide Y as characteristic markers of adaptogenic activity of plant extracts.

ARTICLE INFO

Article history:

Received 10 May 2013

Received in revised form 28 May 2013

Accepted 2 July 2013

Keywords:

Chaperon Hsp72

Neuropeptide Y

Adaptogen

Neuroglia cells

Primary human neurons.

ABSTRACT

We have previously demonstrated that ADAPT-232, a fixed combination of adaptogenic substances derived from Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract stimulated the expression and release of neuropeptide Y (NPY) and molecular chaperone Hsp72 from isolated human neurolgia cells. Both of these mediators of stress response are known to play an important role in regulation of neuroendocrine system and immune response. We further demonstrated that ADAPT-232 induced release of Hsp70 is mediated by NPY, suggesting an existence of NPY-mediated pathway of activation of Hsp72 release into the blood circulation system. The objective of this study was to determine whether this pathway is common for adaptogens and whether NPY and/or Hsp72 can be considered as necessary specific biomarkers for adaptogenic activity. The release of NPY and Hsp72 from neuroglia cells in response to treatment with various plant extracts (n =23) including selected validated adaptogens, partly validated adaptogens, claimed but negligibly validated adaptogens and some other plant extracts affecting neuroendocrine and immune systems but never considered as adaptogens was measured using high throughput ELISA techniques. We demonstrated that adaptogens, e.g. R. rosea, S. chinensis and E. senticosus stimulate both NPY and Hsp70 release from neuroblastoma cells, while tonics and stimulants have no significant effect on NPY in this in vitro test. In the groups of partly validated adaptogens the effect of Panax ginseng and Withania somnifera was not statistically significant both on NPY and Hsp70 release, while the activating effect of Bryonia alba and Rhaponticum cartamoides was significant only on Hsp70. In contrast, all tested non-adaptogens, such as antiinflammatoty plant extracts Matricaria recutita, Pelargonium sidoides. Hedera helix and Vitis vinifera significantly inhibit Hsp70 release and have no influence on NPY release from neuroblastoma cells. These experiments were further validated using primary human neurons and confirmed that adaptogens activate the release of both NPY and Hsp70, while tested non adaptogens were inactive in NPY assay and inhibit the release of Hsp70. Taken together, our data demonstrates for the first time that neuropeptide Y and heat shock protein Hsp70 can be used as molecular biomarkers for adaptogenic activity.

[c] 2013 Elsevier GmbH. All rights reserved.

Introduction

The term adaptogen refers to "metabolic regulators which increase the ability of an organism to adapt to environmental stressors and prevent damage to the organism by such stressors". This definition was suggested and agreed upon at an International Conference on Adaptogens in Gothenburg, Sweden in 1996. The concept is based on the classical theory of stress developed by H. Selye and initially defined as a state of threatened home-osthasis (Selye, 1950; Chrousos and Gold, 1992). Historically, the notion adaptogens (phytoadaptogens) derived from an extensive survey and screening of active substances of botanical origin during 1960-1980 in USSR (Brekhman and Dardymov, 1968; Panossian et al., 1999a).

By 1984, over 1200 publications of pharmacological and clinical studies had been published in Russia/USSR (Brekhman, 1982; Panossian et al., 1999a). Recent pharmacological and clinical studies have shown a high level of consistency with Russian studies (Farnsworth et al., 1985; Davydov and Krikorian, 2000; Huang et al., 2011; Panossian and Wikman, 2008, 2009, 2010; Panossian et al. 2010; Panossian and Wagner, 2005). Several major groups of active substances were identified as active constituents of adaptogenic plants, such as: phenethyl-, phenylpropen- derivatives including lignans, tetracyclic triterpenes and their glycosides, (Brekhman and Dardymov, 1968; Wagner et al., 1994; Panossian et al., 1999a). The adaptogen concept can currently be said to be generally accepted as a category, if not in mainstream pharmacology at least in natural substances pharmacology (EMEA, 2008; EFSA, 2009; Samuelsson and Bohlin, 2009; Saggu and Kumar, 2009; Winston and Maims, 2007; Mashkovskij, 1977; Lazarev, 1958).

A growing interest to stress protective drugs of natural origin has initiated an extensive search of adaptogens all over the world. However, there is a lack of generally accepted standard set of validated methods of the assessment of adaptogenic activity still exists. Regardless of both the kind of system (organism, cell, etc.) or what kind of disturbance to the system, a set of key biomarkers correlating with adaptogenic activity is called for.

A number of recent studies have revealed the important role various mediators of the stress response on the two levels of metabolic regulation by adaptogens:

* Level one: Whole organism--adaptogens support homeostasis and neuroendocrine regulation of hypothalamic-pituitary-adrenal (HPA) axis (Panossian and Wagner, 2011; Panossian, 2003) (Fig. 1). Stress hormones cortisol (Panossian et al., 2007; Olsson et al., 2009), neuropeptide Y (NPY) (Panossian et al., 2012), and other circulating mediators of the stress response such as nitric oxide (Panossian et al., 2007), membrane bound G-protein receptors (Panossian et al., 2013) and molecular chaperons Hsp70 (Panossian et al., 2009, 2012).

[FIGURE 1 OMITTED]

* Level two: Cellular level--adaptogens modulate gene expression (transcriptional control of metabolic regulation) of key mediators of intracellular communications involved in stress induced signal transduction pathways, including G-protein signaling cAMP-mediated pathway (Panossian et al., 2013), G-protein signaling phosphatitylinositol and pholpholipase C pathways (Panossian et al., 2013), stress-activated protein kinase JNIK (MAPK-9) (Panossian et al., 2007), heat shock protein Hsp70 (stress-protective effect on cellular level) (Panossian et al., 2009, 2012), heat shock factor HSF-1 (Panossian et al., 2012), neuropeptide Y (NPY) (Panossian et al., 2012) and forckhead transcription factor FOXO (Wiegant et al., 2009).

These studies and in particular the most recent have revealed the important role of Hsp70 and neuropeptide Y in adaptogenic activity.

Therefore, the main aim of this study was to test the validity of these stress markers as necessary discriminators for an adaptogenic activity. More precisely: a significant increase of both NPY and Hsp70 as a necessary condition for being an adaptogen.

To test this hypothesis it was important select some different groups of other categories for comparison with true (validated) adaptogens.

The main method to be used was measuring the release of NPY and Hsp72 from neuroglia cells in response to treatment with various plant extracts using high throughput ELISA techniques.

A number of frequently used botanical drugs and substances were compared. The selection criteria was mainly to have representatives from four different groups:

* Validated adaptogens: Golden (Arctic) Root, Schisanda, Siberian Ginseng.

* Partly validated adaptogens: Ashwagandha, Maral Root, Bryony, Ginseng.

* Claimed but negligible validated adaptogens: Sea Buckthorn, Basil, Cancer Bush, Licorice, Maca, Astragale, Bastard Ginseng, Co alyceps, Ginkgo.

* Some other more randomly chosen widely used tonics, CNS-and immune active plants with recognized efficacy for their indications: Guarana, Roman Chamomile, Valerian, Chinese Corn-bind, South African Geranium, English Ivy, and Grape.

Glial cells were selected for this study because of several reasons. They are known to contribute to the defence of the brain and maintaining brain homeostasis through several mechanisms including the expression of the innate immune response. Glial cells are know to promote the clearance of neurotoxic proteins and apoptotic cells from the CNS, regulate the entry of inflammatory systemic cells into the brain at the blood brain barrier, stimulate both tissue repair and the rapid restoration of tissue homeostasis and regulate metabolic supply of energy and other substances (Guzhova et al., 2001; Nguyen et al., 2002; Hauwel et al., 2005). In addition, Glial cells are involved in the uptake of neurotransmitters (Henn and Hamberger, 1971), release of neurotrophic factors, modulation of synaptic activity and regulation of neuronal plasticity (Haydon, 2001; Ullian et al., 2001).

Materials and methods

Plant extracts

The extracts of R.rosea L. roots, E. senticosus (Rupr. et Maxim) Harms roots and S. chinensis (Turzc) Baill. berries, were manufactured in Swedish Herbal Institute in accordance to ICH Q7A and EMEA guidelines for Good Agricultural and Collecting Practice (GACP) and Good Manufacturing Practice (GMP) of active pharmaceutical ingredients (API). We have previously described the detailed composition and chemical characterization of ADAPT-232 and ADAPT-232 forte (Panossian et al., 2009, 2012; Aslanyan et al., 2010). All other extracts used in this study were purchased from various manufacturers (Table 1). Working samples used in subsequent experiments were prepared by dilution of 100 pi stock solutions (5 mg/m1) of genuine extracts with 900 [micro]l of phosphate buffered saline solution (PBS). Working solutions of 1000 [micro]l were added to 4 ml of cell culture to obtain the final concentrations of the extracts 125 [micro]g/m1 in incubation media.
Table 1

Solubility of herbal preparations used in the study.

Nr  Extract:      Latin name, type  Analytical marker        Solvents
    Plant name    of extract        compound (c)             used to
    and part                                                 dissolve
    used                                                     the
                                                             extracts

1   Golden        Rhodiola rosea    2.5%--salidroside        40%
    (Arctic)      soft extract                               ethanol
    Root (1)      (a)

2   schisandra    Schisandra        1.4%--schizandrin        40%
    fruit (1)     chinensis soft                             ethanol
                  extract (a)

3   Siberian      Eleutherococcus   1.8%--Eleutherosides B   40%
    Ginseng root  senticosus soft   and E                    ethanol
    (2)           extract (a)

4   Ashwagandha   Withania          2.5%--withanolides,      Water
    root (1)      somnifera soft    2.0%--alkaloids
                  extract (a)       (withasomnin)

5   Maral Root    Rhaponticum       4%--ecdisterones         Water
    (3)           cartamoides soft
                  extract (a)

6   Bryony root   Bryonia alba dry  4.27%--cucurbitacins     Water
    (4)           extract (4)

7   Basil herb    Ocimum sanctum    11.8%--polyphenols       Water
    (5)           soft extract
                  (5)

8   Ginseng root  Panax ginseng     12.5%--ginsenosides      Water
    (6)           dry extract (6)

9   Sea           Hippopbae         33.4%--polyphenols       40%
    Buckthorn     rhamnoides soft                            ethanol
    leaves (2)    extract (a)

10  Ginkgo        Ginkgo biloba     2.37%--flavonglycosides  Water
    leaves (12)   soft extract
                  (12)

11  Cancer Bush   Sutherlandia      16.2%--cycloartanes      Water
    herb (7)      frutesence dry
                  extract (a)

12  Liquorice     Glycyrrhiza       7-9%--glycyrrhizin       Water
    root (8)      glabra dry
                  extract (8)

13  Maca root     Lepidium meyenii  ?%--alkalamides (d)      Water
    (9)           dry extract (a)

14  Astragale     Astragalus        ?%--astragaloside (d)    Water
    roots (2)     membranaceus
                  soft extrac (a)

15  Chinese       Polygonum         ?%--emodin. phiscion,    Water
    Cornbind      multiflorum dry   hydroxy-stylbenes (d)
    (2)           extract (2)

16  Cordyceps     Ophiocordyceps    13.5%--cordicepic acid.  Water
    fungus (10)   sinensis dry      0.5%--adenosine
                  extract (10)

17  Bastard       Codonopsis        ?%--Tangshenoside (d)    Water
    Cinseng,      pilasula dry
    Bellflower    extract (2)
    root (2)

18  Guarana       Paullina cunapa   22%--caffeine            Water
    fruil (11)    dry extract
                  (11)

19  Valerian      Valeriana         0.69%--cyclopentane      40%
    root (12)     officinalis dry   sesquiterpenes           ethanol
                  extract (12)

20  Roman         Matricaria        1.5%--apigenin           40%
    Chamomile     recutita soft     7-glucoside              ethanol
    flowers (1)   extract (a)

21  South         Pelargonium       41.2%--polyphenols       40%
    African       sidoides soft                              ethanol
    Geranium      extract (a)
    roots (7)

22  English Ivy   Hedera helix      12.8%--hederasaponins    Water
    herb (1)      soft extract
                  (a)

23  Grape         Vitis vinifera    25.8%--polyphenols       Water
    crests,       soft extract
    seeds. Husk   (a)
    (13)

Nr  Solubility
        of the
      extracts
           (b)

 1         9.5

 2         6.5

 3        10.0

 4        10.1

 5        21.2

 6        13.0

 7         8.4

 8        15.0

 9        28.7

10        12.5

11        12.5

12        10.0

13         8.0

14        83.3

15        12.0

16        12.0

17        10.0

18        12.0

19        12.5

20         8.2

21        22.0

22        45.2

23        32.2

(1-13.) Supplier of Crude drug or ready extract: 1, Martin
Bauer; 2, Nanchang; 3, Sunytitofarnacia; 4, Arpirnecl; 5,
Farkhi; 6, Indena; 7, Aftiplex; 8, Menozzi; 9, Galke; 10 -
Hangzhou Greensky Biological Tech; 11, Naturex; 12, Fliashman;
13, Puyda.

(a.) Manufacturer of the water alcohol extracts is the Swedish
Herbal Institute.

(b.) Max amount of the extract (dry residue) that is possible
to dissolve in 1 ml of the solvent (mg).

(c.) Content (%) in dry genuine extracts.

(d.) Content of analytical marker compounds is not specified.


Accordingly, two controls were used in the assays, one control contained 0.8% ethanol of incubation media (Table 2).
Table 2

Effect of adaptogens, presumably adaprogens and other plants
extracts on release of NPY and Hsp72 proteins from isolated
human neuroglia cells. (a)

Croups      Therapeutic category         Plant name

                                                              NPY

Validated   Adaptogen                    Rhodiola rosea     +27.6
daptogens                                                   [+ or
                                                           -] 5.4
            Adaptogen                    Schisandra         +24.3
                                         chinensis          [+ or
                                                           -] 4.4
            Adaptogen                    Eleutherococcus    +58.0
                                         senticosus         [+ or
                                                          -] 13.6
                                                             (**)
            Adaptogen                    Adapt 232         +120.2
                                         forte+             [+ or
                                                          -] 33.4
                                                             (**)

Partly      Adaptogen/sedafive           Withania         +9.5 [+
validated                                somnifera          or -]
adaptogens                                                    3.6
            Adaptogen                    Rhaponticum        +14.7
                                         carthamoides       [+ or
                                                           -] 3.7
            Adaptogen/anti-inflammatory  Bryonia alba     +8.8 [+
                                                            or -]
                                                              3.2
            Adaptogen/tonic              Panax ginseng    +5.5 [+
                                                            or -]
                                                              3.6

Claimed as  Antiviral/anti-inflammatory  Hippophae          +10.9
adaptogens                               rhamnoides         [+ or
but                                                        -] 0.9
negligible
validated

            Anti-inflammatory            Glycyrrhiza      -1.0 [+
                                         galbra             or -]
                                                              0.8
            Tonic                        Ocimum sanctum   +9.7 [+
                                                            or -]
                                                              3.2
            Cerebral insufficiency       Ginkgo biloba    +2.1 [+
                                                            or -]
                                                              0.9
            Tonic                        Sutherlandia     +1.7 [+
                                         frutessence        or -]
                                                              0.8
            Tonic/Aphrodisiac            Lepidium         -2.5 [+
                                         meyenii            or -]
                                                              1.0
            Tonic/Aphrodisiac            Ophiocordiceps   -0.4 [+
                                         sinensis           or -]
                                                                0
            Tonic                        Codonopsis       -3.3 [+
                                         pilosula           or -]
                                                              1.1
            Stimulant, tonic             Paullina cupana  -1.9 [+
                                                            or -]
                                                              1.2
            Tonic/immune stimulant       Astragalus       -3.5 [+
                                         membranaceus       or -]
                                                              1.4

Other       Tonic                        Polygonum        -1.5 [+
drugs                                    multiflorum        or -]
                                                              0.8
            Anti-inflammatory/sedative   Matricaria       +7.0 [+
                                         recutita           or -]
                                                              2.1
            Sedative                     Valeriana        +3.7 [+
                                         officinalis        or -]
                                                              1.7
            Antibiotic/ immune           Pelargonium      -2.5 [+
            stimulant                    sidoides           or -]
                                                              1.3
            Spasmolytic                  Hedera helix     -2.5 [+
                                                            or -]
                                                              1.3
            Antioxidant                  Vitis vinifera   -3.9 [+
                                                            or -]
                                                              1.3

Croups      % increased
            release (+)
               [+ or -]
               SEM or %
              decreased
            release (-)
               [+ or -]
             SEM of the
             respective
               proteins

                  Hsp72

Validated   +38.4 [+ or
adaptogens       -] 4.4
                   (**)
            +40.6 [+ or
                 -] 1.1
                   (**)
            +34.8 [+ or
                 -] 2.8
                   (**)
            +44.4 [+ or
                 -] 3.6
                   (**)

Partly      +13.4 [+ or
validated        -] 1.8
adaptogens
            +27.4 [+ or
             -] 43 (**)
            +32.8 [+ or
                 -] 2.4
                   (**)
            +15.0 [+ or
                 -] 6.7

Claimed as   56.0 [+ or
adaptogens       -] 6.9
but                (**)
negligible
validated
            -12.2 [+ or
                 -] 2.1
            -21.0 [+ or
                 -] 1.9
            -11.7 [+ or
                 -] 1.7
             -273 [+ or
                 -] 1.9
                   (**)
            -19.0 [+ or
                 -] 3.8
            +24.8 [+ or
                 -] 8.4
                   (**)
            +22.7 [+ or
             -] 0.3 (*)
            +35.5 [+ or
             -] 4.8(**)
            -12.4 [+ or
                 -] 1.6

Other       -32.7 [+ or
drugs            -] 3.4
                   (**)
            -19.2 [+ or
                 -] 4.6
            +46.4 [+ or
                 -] 7.4
                   (**)
            -62.1 [+ or
                 -] 0.5
                   (**)
            -44.2 [+ or
                 -] 2.3
                   (**)
            -24.3 [+ or
             -] 2.9 (*)

Statistically significant data are in bold.

(a.) Results are percentage (%) increases in concentration of
NPY and Hsp72 out of four independently performed experiments
for each protein using T89G human neuroglia cells.

(*.) p <0.05 vs to control group in one way analysis of
variances, with Dunnett's multiple comparison test.

(**.) p <0.01 vs to control group in one way analysis
of variances, with Dunnett's multiple comparison test.


Cells, cell lines and culture conditions

The human neuroglia cell line T98G (ATCC, CRL-1690; Manassas, VA) has a hyperpentaploid chromosome count that was derived from a 61-year-old Caucasian male with glioblastoma multiforme. Human neuroglia T98G cells were cultured from ATCC-formulated Eagle's Minimum Essential Medium (Catalog No. 30-2003) with fetal bovine serum to a final concentration of 10%, 100 U/m1 penicillin, 100 [micro]g/ml streptomycin (ICN, Aurora, Ohio, USA). To avoid stress induced by cell overgrowth, cultures were maintained in a 37[degrees]C-incubator in humidified air with 5% [CO.sub.2] atmosphere. Cells were maintained at a density of 2 x [10.sup.5] cells/ml and passaged with fresh complete medium every 3-4 days. Cell viability was assessed using trypan blue exclusion test and routinely found to contain <5% dead cells.

Primary human neurons were purchased from Neuromics (Edina, MN). Neural tissue was micro-dissected from distinct brain regions. This tissue was shipped live, not frozen, in a nutrient rich medium that keeps the cells viable for weeks. On arrival the cells appear initially round and without processes (Fig. 2A). After 5 days of culture the cells start to extend neuritis with extensive processes (Fig. 2B).

[FIGURE 2 OMITTED]

Briefly, cultures were treated with 4 mg papain (supplemented with 0.07 mM [beta]-mercaptoethanol, 1.1 mM EDTA, 5.5 mM cysteine-HCI) for 30 min at room temperature in 35mm dishes treated overnight with poly-lysine according to the manufactures instructions. After the enzyme treatment, cells were homogenized in the papain solution. The homogenate was then centrifuged (1500 rpm for 2 min). The supernatant was recovered and supplemented with tissue media and homogenized again to produce a single cell suspension, centrifuged again and pellet resuspended in Neurobasal media. Primary human neurons were then plated and used in the subsequent experiments.

Determination of cell viability

Cell viability was determined using the lactate dehydrogenase (LDH) assay. LDH is a stable cytosolic enzyme that is released upon cell lysis. LDH released into cell culture media by dead cells and total LDH contained in living cells was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay according to the manufacturer's instructions (Promega, Madison, WI). Briefly, after various treatment protocols, culture medium (500 11,1) was removed and the remaining cells lysed by adding 500 [micro]l of 5% Triton X-100 solution. After 30 min at room temperature, cell lysate was recovered and incubated for an additional 30 min in the dark with a buffer containing NAD, lactate and tetrazolium. LDH converts lactate to pyruvate, generating NADH, which reduces tetrazolium (yellow) to formazan (red), which is detected by fluorescence (490 nm). LDH release, a marker for cell death was expressed as a percentage of the LDH in the medium over the total LDH (lysate).

Hsp72 enzyme immunoassay

After treatment the human neuroglia cell line T98G was centrifuged to discard floating cells and cellular debris and the total protein content was determined by Bradford analysis using bovine serum albumin as a standard. The supernatant was aliquoted and treated with or without 1% Triton X-100 or 1% Lubrol WX or 0.5% Brij 98 for 10 min at 4[degrees]C with gentle rocking and Hsp72 content measured by standard sandwich ELISA. Briefly, 96-well microtitre plates (Nunc Immunoplate Maxisorp; Life Technologies) were coated with murine monoclonal anti-human Hsp72 (clone C92F3A-5; Stressgen) in carbonate buffer, pH 9.5 (2 p.g/m1) overnight at 4[degrees]C. Plates were then washed with PBS containing 1% Tween-20 (PBS-T) and blocked by incubation with 1% bovine serum albumin in PBS-T. Supernatant was added and bound Hsp72 was detected by the addition of rabbit polyclonal anti-Hsp72 antibody (SPA-812; Stressgen). Bound polyclonal antibody was detected with alkaline phosphatase-conjugated murine monoclonal antibody to rabbit immunoglobulins (Sigma Chemical Co), followed by p-nitrophenyl phosphate substrate (Sigma Chemical Co). The resultant absorbance was measured at 405 nm with a Bio-Rad Benmark Plus plate reader. Standard dose-response curves were generated in parallel with Hsp72 (0 to 20,000 ng/m1; Stressgen), and the concentrations of Hsp72 were determined by reference to these standard curves with ASSAYZAP data analysis software (BIOSOFT). The interassay variability of the Hsp72 immunoassays was <10%.

NPY enzyme immunoassay

Human neuroglia cells were grown to 75% confluence. Twenty-four hours prior to measuring NPY release, cell culture medium was replaced with serum-free DMEM, then incubated with ADAPT-232, or salidroside, or exposed to heat shock (41 [degrees]C, 60 min) and incubated for 24h in a 37[degrees]C incubator. Supernatant was recovered in quadruplicates, centrifuged to clear cellular debris and NPY concentration was measured using the NPY enzyme immunoassay kit (assay sensitivity of 0.09 ng/m1) according to the manufacturer's instructions (Phoenix Pharmaceuticals, CA). Briefly, cleared supernatant was added to 96-well plates containing primary antibody and biotinylated peptide and incubated at room temperature for 2 h. Plates were washed and SA-HRP solution was added to the well and incubated for a further 1 h at room temperature. After washing the plates TMB substrate was added and incubated for an additional 1 h at room temperature. The test was terminated by adding 2 N HCl to the wells. The resultant absorbance was measured at 450 nm with a Bio-Rad Benmark Plus plate reader. The concentration of NPY was determined by reference to these standard curves with ASSAYZAP data analysis software (BIOSOFT). The interassay variability of the NPY enzyme immunoassays was <10%.

Statistical analysis

Data management and calculations of mean values of measurements (in triplicates) from four independent experiments were performed using GraphPad (San Diego, CA, USA) Prism software (version 3.03 for Windows). One-way analysis of variances, with Dunnett's multiple comparison test has been applied for evaluation of p-values and significance of difference of measured variables compared to control.

Results

We demonstrated that validated adaptogens significantly stimulate NPY release from human neuroglia cells (Table 2). Adaptogens stimulate both NPY and Hsp70 release from neuroblastoma cells, while tonics and stimulants have no significant effect on NPY in this in vitro test. In the groups of partly validated adaptogens the effect of Panax ginseng and Withania somnifera was not statistically significant both on NPY and Hsp70 release, while the activating effect of Bryonia alba and Rhaponticum cartamoides extracts was significant only on Hsp70. In contrast, all tested non-adaptogens, have no influence on NPY release from neuroglia cells and significantly inhibit Hsp70 release (anti-inflammatory plant extracts Matricaria recutita, Pelargonium sidoides, Hedera helix, and Vitis vinifera), except of Valerian, which activates the release of Hsp70 (Table 2).

We further demonstrated that adaptogens activate the release both NPY and Hsp70 from freshly harvested primary human neurons. However, non adaptogens are inactive or inhibits release of Hsp70 (Table 3).

Discussion

The evaluation of the adaptogenic activity of plant extracts is a complicated task because of several reasons. One reason is that it is dependent on the definition of adaptogens, which was originally based on a complex physiological condition of an entire organism--the stress, a threatened state of homeosthasis, but not on a single kind of disorder. Therefore, it is not surprising that many mediators of the stress response can be involved in the various interactions with adaptogens on various levels of the regulation of homeostasis:

* The level of a small physiologically active molecules, e.g. cAMP, cortisol, nitric oxide etc.--the so-called metabolomic level of regulation.

* The level of proteins involved in the synthesis or degradation of small molecules and their receptors--the so-called proteomic level of regulation.

* The level of genes encoding the expression of proteins--the so-called genomic and transcriptional level of regulation.

* The level of regulatory systems of an entire organism, e.g. the neuro-endocrine--immune system.

The choice to evaluate the relevance of NPY and Hsp70 in adaptogenic activity was justified in our recent publication (Panossian et al.. 2012) as discussed below. The mechanism of stress-protective action of adaptogens is partially associated with the hypothalamic-pituitary-adrenal (FIPA) axis, a part of the stress system that also contributes to the nervous, cardiovascular, immune, gastrointestinal and endocrine systems (Panossian et al., 1999a). Adaptogens activates the expression of Neuropeptide Y brain cells (Panossian et al., 2012), which is known to activate the release of corticotrophin releasing hormone (CRH) (Haas and George, 1989). This stress-mimetic effect of adaptogens (Panossian et al., 1999b; Wiegant et al., 2009) stimulates innate defence systems, including lisp70, plays an important role in the reparation of stress-induced damages of proteins and recovery of their cellular functions (Panossian et al., 2012). These molecular chaperones promote the correct three-dimensional folding of other proteins, prevents their aggregation and assists in the re-folding of misfolded proteins, which are the main contributors to many devastating human diseases. Hsp72 plays a central role in the mechanism that rids the cell of stress induced misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular functions, thereby playing a critical role in maintaining cellular homeostasis and in protecting cells from stressful conditions and increase cell survival in the face of otherwise lethal cellular stress. In addition, Hsp72 may function as an endogenous 'danger signal' for the immune system (Asea, 2007). Exposure to physical or psychological acute stressors stimulate the release of endogenous Hsp72 into the blood and that elevated Hsp72 functions to facilitate innate immunity in the presence of a bacterial challenge (Asea, 2005; Febbraio and Koukoulas 2000; Fleshner and Johnson, 2005; Gonzalez and Manso, 2004; Johnson and Fleshner, 2005; Lancaster and Febbraio, 2005; Whitham and Fortes, 2008).

We have previously demonstrated that ADAPT-232, a fixed combination of adaptogenic substances derived from Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract SHR-5, and its active constituent salidroside stimulated the expression of NPY and Hsp72 in isolated human neurolgia cells (Panossian et al., 2012). We further validated the central role of NPY in experiments in which pre-treatment of human neuroglia cells with silencing RNA NPY-siRNA and HSF1-siRNA resulted in the significant suppression of ADAPT-232-induced NPY and Hsp72 release. Our studies suggest that the stimulation and release of the stress hormones, NPY and lisp72, into systemic circulation is an innate defence response against mild stressors (ADAPT-232), which increase tolerance and adaptation to stress (Fig. 1).

The objective of this study was to determine whether NPY and/or Hsp72 release is a specific and necessary function of adaptogens. This was achieved by measuring the release of NPY and Hsp72 from neuroglia cells in response to treatment with test compounds including selected validated adaptogens, partly validated adaptogens, claimed but negligibly validated adaptogens and other plant extracts affecting neuroendocrine and immune systems which have never been considered as adaptogens using high throughput ELISA techniques. To determine which test compounds retain the ability to potently stimulate the release of NPY and Hsp72 in a similar fashion as ADAPT-232, we used the experimental protocol described in detail in the Materials and Methods section and as published previously (Panossian et al., 2012). Briefly, we used T98G cells as our target cell line. The T89G cell line is a transformed human neuroglia derived from a 61-year-old Caucasian male with glioblastoma multiforme, which was purchased from ATCC (Manassas, VA). T89G cells were grown to approximately 75% confluency and then treated for 24h with the test compounds and supernatant was extracted and probed for the concentration of released NPY and Hsp72 using ELISA technique. The data presented in this study demonstrate that activation both NPY and Hsp70 is specific for adaptogens (Table 2). Our data suggest that the activation of Hsp70 by Valerian. Guarana, Cordiceps and Codonopsis might be due to a NPY-inclependent mechanism. Our hypothesis suggests that the release of Hsp72 by adaptogens take place via a mechanism dependent on the upregulation of NPY, which is upstream of Hsp72 and all other mediators of stress response involved in effects of adaptogens (Panossian et al., 2012).

It is particularly interesting that the plants, which traditionally have been classified as tonics, like Polygonum multiflorum and Astragalus membranus and stimulants as Paullina clearly differs from the adaptogens. The point here is that these two categories i.e. tonics and stimulants are already being confused for adaptogens.

To determine whether the results we obtained using the transformed human neuroglia cells in which the release of NPY and/or Hsp72 is relevant for physiologically normal cells and evaluation of effects of adaptogens, we repeated the experiments using a system more closely related to the human system; non-transformed human neurons. In these experiments, we selected a few candidate adaptogens and non-adaptogens and admixed then onto freshly harvested primary human neurons, and measured the release of NPY and Hsp72. The validation of the results obtained on neuroblastome cells by experiments freshly harvested primary human neurons demonstrates that adaptogens activate the release both NPY and Hsp70, while non adaptogens are inactive or inhibits release of Hsp70 (Table 3).
Table 3 Treatment of human neurons with select adaptogens results
in significant increased release of NPY and Hsp72 proteins. (a)

Classification  Plant name                         % increased
                                             release (+) [+ or
                                                   -] SEM or %
                                             decreased release
                                               (-) [+ or -]SEM
                                             of the respective
                                                      proteins

                                        NPY              Hsp72

Validated       Rhodiola        +47.2 [+ or     +44.6 [+ or -]
adaptogens      rosea radix     -] 13.1 (*)           3.0 (**)
                soft extract
                Adapt 232      +183.3 [+ or     +84.2 [+ or -]
                forte+         -] 25.2 (**)           3.0 (**)

Negligible      Sutherlandia  +1.6 [+ or -]      -3.5 [+ or -]
validated       frutessence             1.0                0.8
adaptogens      herba dry
                extract
                Glycyrrhiza    -1.9[+ or -]     -10.8 [+ or -]
                galbra radix            1.0                1.9
                dry extract

Other drugs     Pelargonium   -4.2 [+ or -]     -34.3 [+ or -]
                sidoides                0.9           12.2 (*)
                radix soft
                extract
                Hedera helix  -4.2 [+ or -]     -34.2 [+ or -]
                herba soft              0.9           12.2 (*)
                extract

Statistically significant data are in bold.

(a.) Results are percentage (%) increases in concentration of
NPY and Hsp72 in triplicates and is a representative
experiment from three independently performed experiments
using freshly harvested primary human neurons purchased from
Nuromics (Edina, MN).

(*.) p<0.05, vs to control group in one way analysis of
variances, with Dunnett's multiple comparison test.

(**.) p <0.01, vs to control group in one way analysis of
variances, with Dunnett's multiple comparison test.


Our data supports the proposition that stimulation and release of the stress hormones, NPY and Hsp70 into the blood circulating system is an innate defence response to mild stressors (adaptogens), which increases tolerance and adaptation to stress. This gives rise to adaptive and stress-protective effects via various components of central nervous, sympathetic, endocrine, immune, cardiovascular and gastrointestinal systems.

The released Hsp70 acts as an endogenous danger signal and plays an important role in immune stimulation. While released NPY plays a major role on the HPA axis and maintaining energy balance. Both mediators are directly involved in adaptation to stress, resulting in increased survival, longevity and cognitive function.

Taken together, our studies demonstrate that NPY and Hsp70 assays in isolated neuroglia cells might be useful for the assessment of adaptogenic activity of medicinal plants. Stimulation of both NPY and Hsp70 release is necessary but perhaps not a sufficient requirement for adaptogenic activity that has to be supported by clinical studies.

Conflicts of interest

P.K., A.A., and A.P. declare no competing financial interests. G.W. is a stockholder in the Swedish Herbal Institute (SHI).

* Corresponding author. Tel.: +14048448253.

** Corresponding author. Tel.: +46702818171.

E-mail addresses: aasea@msm.edu (A. Asea).ap@shi.se (A. Panossian).

0944-71131$--see front matter [c] 2013 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.phymed.2013.07.001

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Alexzander Asea (b), *, Punit Kaur (b), Alexander Panossian (a), **, Karl Georg Wikman (a)

(a) Swedish Herbal Institute, Research and Development, kovlingevagen 21, SE-312 75 Vallberga, Sweden

(b) Morehouse School of Medicine, Department of Microbiology, Biochemistry and Immunology & Department of Pathology and Laboratory Medicine, 720 Westview Drive SW, Atlanta, GA 30310-1495, USA
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Author:Asea, Alexzander; Kaur, Punit; Panossian, Alexander; Wikman, Karl Georg
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Nov 15, 2013
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