**4.1 Alkaloids**

Radioligand binding assay using [35S] TBPS and [3 H] flunitrazepam analyzed the weak partial agonistic activity of Colchicine and (−) cornigerine along with six other colchicinoids from *Colchicum brachyphyllum*. These two molecules displayed 25% of the action of 10 μM allopregnanolone, but the (−) activity of colchicines was none. (−) colchicines acted as a GABA competitive antagonis [32, 33]. GABA modulation is also recorded in proto alkaloid leonurine, belonging to an East Asian herb called *Leonurus japonicus*, indicated for anxiety, depression, nervousness, and insomnia. The molecule showed half-maximal inhibitory concentration (IC50) values of 15 μg/mL and 123 μg/mL, respectively in a radio ligand assay with [3 H] gabazine and [3 H] flumazenil [34].

Piperine and piperanine belonging to the class of piperidine-alkaloids were investigated in the immature egg cell of *Xenopus laevis*. The binding site of the molecule was confirmed to be a benzodiazepine binding site as Flumazenil (5 mg/kg) [35].

A β-carboline named annomontine also shows GABA dependant activity which was separated from the plant *Annona purpurea* [36]. In the EPM test, the compound increased the time spent in the open arms and the open arm entries at 10 and 30 mg/kg, but not the total arm entries. These effects are controlled via the benzodiazepine binding site as it was confirmed with antagonist Flumazenil at a dose of 3 mg/kg. Another alkaloid, a non-competitive GABA(A) receptor antagonist is diterpene alkaloid and this was separated from the plant *Aconitum leucostomum* having an IC50 19.6 μM. Radioligand studies with the help of [3 H] muscimol produced an IC50 value of 7.06 μM and a Kd value of 6.31 nM. Specific binding site specificity was shown by β-carbolines and picroacridine alkaloids as they bind to the benzodiazepine binding site Benzodiazepine binding sites can at least be excluded for piperidine alkaloids and protoberberine alkaloids. This holds for songorine in GABA/muscimol binding site as well [37].

Three colchicinoids displayed unspecific binding with weak action on both benzodiazepine and TBPS/bicuculline binding sites. Colchicine is the antagonist, but androbiphenyline and cornigerine are partial agonists. Protoalkaloid Leonurine shows binding to various sites, with decreased affinities to, GABA/muscimol and the benzodiazepine binding site. Protoberberine type 2 alkaloids were able to modulate GABA(A) receptors, but unsaturated type 1 alkaloids displayed no effects.

#### **4.2 Alkanes**

The odor substance, 1-octen-3-ol is part of the GABAA sensory receptor modification research and has a stimulation rate of 295 ± 50 percent at a particular concentration of 300 μM and 1 μM GABA [52]. Ceramide (N-[(2S,3R,4E,6E)-1,3 dihydroxyhenicose-4,6-dien-2-yl] tridecanamide) separated from the Red Sea soft coral *Sarcophytum auritum.* This works as GABA agonist and produce anxiolytic effect in animal models evaluated by EPM test in which the animal spent more time in open arms and time spent in light in light/dark test whereas all its action can be altered by the action of bicuculline (GABA antagonist) [38, 52].

Two polyacetylenes extracted from *Oenanthe fistulosa,* Oenanthotoxine and dihydroenanthotoxine, provided major inhibitory activity on GABA receptors with IC50 values of 0.835 μM and 1.29 μM respectively on GABA (A) receptors The potential explanation for the indications which include water drop worm intoxication (Oenanthe crocata) and facial muscle contractions which is due to the inhibition of GABAergic reactions. The substance that allosterically regulate GABAbinding, non-competitively inhibits ion channel and also eradicates the desensitization of the receptor was constituted by modes of action of oenanthotoxin [39].

At a very low concentration the component falcarindiol obtained from *Oenanthe crocata* effectively regulates GABAergic currents. This component at low concentration (1 μM for falcarindiol and 0.1 μM for falcarinol) promotes the ion currents caused by GABA, on the other hand at higher concentration it inhibits the action of GABA. Moreover, the sedative but not convulsive result in animals is triggered by the low-dose effect, whereas the large doses in insect herbivores act as insecticides [40].

The three polyacetylenes MS-1, MS-2, and MS-4 were obtained from *Cussonia zimmermannii* with recorded GABA(A) modifying activity [41].

However, the potency and/or affinity were demonstrated in the small micro molar range, but that varies significantly in terms of toxicity. Two structural characteristics (groups of allyl and terminal hydroxyl) that are present in five (most) poisonous natural products produced toxicity. It suggests that the terminal hydroxyl class is vital for the toxicity. Further, both the oenanthotoxins and dihydrooenanthotoxins require the allyl hydroxyl group but are highly toxic. On the other hand falcarinol and falcarindiol, which have an allylic class but not the final hydroxy group, showed decreased toxicity. None of the two "toxic characteristics" are present in the last three polyacetylenes group and are also not documented to display inhibitory behavior consistent with this theory. It would be necessary to investigate whether hydrolyzation has led to GABA (A) receptor antagonism because MS-4 has a terminal acetyloxy-group [38, 52].

#### **4.3 Flavones**

The substance Oroxylin A, allosterically to block GABA-mediated receptor by its action on chloride currents, and thus it describes the results of a previous in vivo study in which the substance exhibited antagonistic diazepam-induced effects [42, 43].

Wogonin was considered for the induction of GABA-induced chloride currents by using electrophysiological methods where it shown a stimulation of 57% at a concentration of 30 μM in the presence of 1 μM GABA where at 3 μM half maximal stimulation was noticed. It was also tested pharmacologically at a dose of 7.5, 15 and 30 mg/kg by using Elevated plus maze and hole board test. The wogonin showed anxiolytic effects. These data recommend that wogonin yielded anxiolytic by positive allosteric modulation of the GABAA receptor complex through benzodiazepine site interaction [43].

The chrysin is from *Scutellaria baicalensis* class which was separated from *Passiflora caerulea* [44, 63]. Chrysin was testified as partial agonist of central benzodiazepine receptors which reduced anxiety and does not induced sedative and muscle relaxation. The pharmacological effect of chrysin was observed in mice at 1 mg/kg in Elevated plus maze test. The anxiolytic effect was observed by increasing the number of entries and time spent in the open arm. The horizontal wire test showed a decreased percentage of animals grasping the horizontal wire, while in the hole board test an increase in time spent head-dipping at 3 mg/kg was observed, but no sedative effects at doses of 3 and 6 mg/kg [44].

Flavone compounds like wogoninnn and chrysin shows diazepam like anxiolytic effect whereas Oroxylin A antagonizing the effects provoked by diazepam.

## **4.4 Flavanes**

(S) naringenin was isolated from the ethanol extract of *leaves Mentha aquatica* and evaluated against [3 H] flumazenil which exhibits an IC50 value of 26 mM. This compound can effectively modulate GABA function [45].

Glabrol, is the prenylated flavanone its three Diels-Alder type derivatives, sanggenon C, D, and G and were obtained from the root extract of *Morusalba*. All three molecules, with EC50 values in the range from 13.4 to 16.7 μM, increased chloride-induced GABA by over 700 percent (100 μM) [46].

In particular, two 8-lavandulyl-flavanones produced GABA-induced chloride impulses to potentiate by about 600 percent compared to the third 8-lavandulylflavanonol which is substantially less active.

The compounds like (S) naringenin, glabrol and 8-lavandulyl-flavanones acts at benzodiazepine site of GABA receptor which was analyzed using radio ligand binding assay.

#### **4.5 Isoflavanoids and chalcones**

*Adenocarpus cinncinatus* considered as the source of 2′, 4′, 7-trihydroxy-8-(3-methylbut-2-en-1-yl) isoflavone. Its stimulatory effect exhibits a uplift of GABA-induced chloride currents [64]. At a concentration of 30 nM, the substance increased GABA-induced chloride currents by 135 percent with a maximum potentiation of 581 percent at a level of 100 μM.

Isoliquiritigenin increased GABA-induced currents by of 151% at a dose of 10 M with a patch-clamp method on dorsal raphe neurons [48].

The *Sophora flavescens lavandulyl chalcone* is Kuraridine, which potentiates GABA-induced chloride currents by 719.7 percent at a dose of 10 M with a maximal activation rate of 891.5 percent [56].

The findings for isoflavonoids and chalcones are consistent with the results of the last two sections: isoflavone genistein blocks chloride currents in the same way as its flavone equivalents apigenin. The binding of [3 H] flunitrazepam inhibits chalcone isoliquiritigenin, furthermore the prenylated types show a marked ability of more than 500 percent (95.97) to around 900 percent.

In these compounds the substitution of one hydroxy and one methoxy group in both aromatic rings shows better potency. Overall, all of these compounds shown GABA (A) receptor agonist type action.

#### **4.6 Terpenes**

#### *4.6.1 Monoterpenes*

(+) borneol, (−) borneol, (−) bornyl acetate, is borneol, and camphor acting on GABA(A) receptors which were stated in *oocytes of Xenopus laevis*. With the increased stimulation reported for (+)-borneol and (−)-borneol, all other substances resulted in a marked maximum potentiation of GABA-induced chloride currents. EC50 values were, however, in the large micromolar range with the smallest score reported for bornyl acetate (111.2 μM) [49, 65].

In a radioligand binding assay measured on α and β thujone against [3 H] EBOB, where the substances displayed IC50 values of 13 and 37 μM. The β-thujone was identified as a non-competitive antagonist with an IC50 value of 21 μM in additional electrophysiological studies. Studies have confirmed these molecules acts by allosteric decrease of GABA-induced chloride currents. α-thujone has been reported in a survey on GABAergic miniature inhibitory currents to decrease their frequency and amplitude and to moderately influence their kinetics. The study concluded that alpha-thujone had gating receptor activity as this substance decreased the amplitude of current reactions to exogenous GABA and influenced their initiation, desensitization, and neutralization [50]. Epoxy-carvone was studied using MES, PTZ, and picrotoxin-induced seizure models for its anticonvulsant properties [66].

In *Xenopus oocytes*, thymol an aromatic monoterpene is known from a variety of Thymus species, was examined on α<sup>1</sup> β<sup>2</sup> γ3 where chloride-induced GABA-currents increased by 416% at a concentration of 100 μM [51].

*Isopulegol* has been tested in-vivo for its anxiolytic ability. In the hole board test and Elevated plus maze [EPM] test at a concentration of 25 and 50 mg/kg, the isopulegol has been shown to raise the number of head dips in the hole board test which specifies anxiolytic effect in which the number of open arm entries along with the time spent in the arm was also increased in EPM test. In the EPM test of isopulegol results reduced the animal's aversion to the open arms as well as promoted the exploration which specifies anxiolytic effect [67].

In an anxiolytic-like behavioral study, the (+)-limonene epoxide at various doses of 25, 50, and 75 mg/kg showed an improvement in open arms inputs and time spent in open arms in the EPM test and decrease in the number of crossing, grooming, and rearing is found in the open field test, further implying the sedative effects of the drug [55]. The anxiolytic effect was reported by a follow-up study in which the compound demonstrated a decrease in the number of buried marbles in the buried marble test at a dose of 25, 50, and 75 mg/kg [68]. In several studies, Carvacryl acetate was also tested for anxiolytic and sedative effects. The EPM test shows that the compound increased the number of open arm entries at a dose of 100 mg/kg and the time spent in the open arm at doses from 25 to 100 mg/kg. In case of the light/dark test it increased the number and time spent in the light area at doses from 25 to 100 mg/kg. In the buried marbles test reduction of buried marbles number was observed at doses from 25 to 100 mg/kg, but no co-ordination impairment in the Rotarod test and no decrease in locomotor activity is observed in the open field test were measured at the same doses [69].

A few monoterpenes have been studied for their GABA receptor modulation action and the highest potential of chloride channel opening was observed for bicyclic alocohols, like (+) and (−)-borneol whereas isoborneol showed distinct potentiation. Oxidation of the hydroxy-group or the presence of an exocyclic methylene group causes decrease in the activity. The only monocyclic monoterpenes positive receptor modulation was observed by thymol.

#### *4.6.2 Sesquiterpenes*

Two monoterpenoid moieties namely α caryophyllene and β caryophyllene belonging to *Sideritis sp*. that displayed medium modulation of GABA-mediated chloride channels (117 and 115%, respectively).

Curdione and curcumol were extracted from the oil of *Curcumaerhizoma* and were tested on GABAA receptors expressed. The molecules increased GABAmediated chloride channel activity with 133 and 175.7%, respectively at a concentration of 50 μM. The EC50 value of Curcumol was found to be 34.4 μM and the highest activity of 251% was found at 300 μM [53].

The highest induction of GABA-mediated chloride channel of around 400% was found in (+) cuparenol and (+)-dihydrocuparenic acid.

At 300 μM, when Atractylenoids I, II and III from *Atractylodesmacrocephala* was tested on GABA (A) receptors highest stimulation of 96 to 166% was observed with an EC50 value of 12, 70 and 99 μM, respectively [54, 70].

Anisatin is oxygenated sesquiterpene lactone separated from *Illicium anisatum* is a potent noncompetitive antagonist of GABAA receptor that has an activity similar to picrotoxin [71]. Studies demonstrated that anisatin at 1 μM decreased chloride currents created by 30 μM GABA to 41.7%. The IC50 value was measured with 1.10 μM along with an IC50 value of 0.42 μM for picrotoxinin, which is the active compound of picrotoxin was obtained. An indication that anisatin binds to the picrostatin site of the receptor was shown in a radioligand binding assay with an IC50 value of 0.43 μM against [3 H] EBOB. One very potent sesquiterpene is xenovulene A, which was separated from the fungus Acremonium striatum (now classified as Sarocladium striatum) [72].

As a result of the structural differences of the sesquiterpenes only restricted conclusions on their structure–activity relationship can be drawn. Reduction of the acidic function to an alcoholic function does not change the activity whereas the change of the isopropenyl-function of compound to a plane isopropanyl-moiety leads to a significant loss of activity.

#### *4.6.3 Diterpenes*

In this Section 14 diterpenes which are having the actions on GABA are discussed. Miltirone, a Salvia miltiorrhiza tanshinone, was assessed against [3 H] flunitrazepam with an IC50 value of 0.3 M in a radioligand-binding analysis [73].

Dehydroabietic acid has been segregated and examined in *Xenopus laevisocytes* from *Boswellia thurifera*, now known as *Boswellia serrata* [47]. GABA-induced chloride currents were enhanced by the substance by 397.5 percent at 100 μM and displayed an EC50 value of 8.7 μM. Isopimaric and sandaropimaric acid were extracted and examined from *Biota Orientalis*, currently known as Platycladusorientalis, in the Xenopus oocyte assay [74]. The substances showed a maximum stimulation effect of 425.2 and 855.7 percent of GABA-induced chloride currents at 500 μM and EC50 values of 141.6 and 33.2 μM, respectively.

Two diterpenes of phyllocladane namely 17-dihydroxyphyllocladane-3-one and 16,17,18-trihydroxyphyllocladane-3-one types were obtained from *Aloysia virgata* and assessed for GABA(A) affinity to [3 H] flumazenil with inhibitory constant [Ki] of 111 and 56 μM. Both compounds were studied in vivo, with compound, 17-dihydroxyphyllocladane-3-one which exhibits increased locomotor activity at a dose of 1 mg/kg in the locomotor activity test and increased rearing at 0.3 and 1 mg/kg in the hole board test. Compound 16,17,18-trihydroxyphyllocladane-3-one increased the number of head dips at 0.3 and 3 mg/kg, the number of rears at a dose of 1 mg/ kg and the time spent head-dipping at a dose of 3 mg/kg. The compound at a dose

of 1 mg/kg increased the number of open arm entries in the EPM test and the time spent in the light area as well as the number of transitions in the light/dark test [75].

Two diterpenes of type labdane, cerumin A and coronarin D, were obtained from Curcuma kwangsiensis [57]. In the Xenopus oocyte assay, substances at a 300 M concentration stimulated GABA-induced chloride currents by 309.4 and 211.0 percent, with EC50 values of 24.9 and 35.7 M. Ginkgolides A B and C which are diterpene trilactones of *Ginkgo biloba*, are moderately active GABAA receptor antagonists with Ki values of 14.5, 12.7 and 16.3 M in *Xenopus laevis oocytes* [58].

Some results suggest that compounds like 7-methoxyrosmanol and galdosol increases 10-fold receptor affinity by an oxo-group at 7nth position instead of methoxy group. On the other hand, for compounds isopimaric acid and sandaropimaric acid, the change from the 7th to the 8th position of the double bond and thus to the C-ring of the substance doubles the maximum stimulatory effects and significantly decreases the EC50 value There are no clear variations in the inhibitory action of bilobalide and ginkgolide A-C in their IC50 values or in their ability to inhibit chloride current induced by GABA. Therefore, all these diterpenes works as GABA receptor agonists which help in chloride current flow.

#### *4.6.4 Triterpenes*

Asiatic acid was separated from *Centella Asiatica* and its anxiolytic effects were analyzed in the EPM test. The compound displayed no action on the open arm time but reduced the motile time and the highest speed at 30 mg/kg. These actions were blocked by flumazenil [76].

Ginsenoside C, is a glycoside isolated from *Panax ginseng* was tested on GABA(A) receptors expressed in xenopus laevis oocytes which were found to potentiate GABA-induced chloride currents with an EC50 value of 53.2 μM [77].

Four cycloartane glycosides actein, cimigenol-3-O-β-D-xylopyranoside 25-O-acetylcimigenol-3-O-α-L-arabinopyranoside, 23-O-acetylshengmanol-3- O-β D-xylopyranoside were extracted from *Actaea racemosa* root systems (black cohosh) and assessed in *Xenopus laevis* oocytes for their capacity for GABA-induced chloride currents [74]. Substances like actein, cimigenol-3-O-β-D-xylopyranoside and 25-O-acetylcimigenol-3-O-α-L-arabinopyranoside which are isolated constituents from rhizomes of Actaea rasemosa exhibited potentiation of GABA-induced chloride currents in the range of 256 to 378 percent at a concentration of 300 M, while 23-O-acetylshengmanol-3-O-D-xylopyranosides reported stimulation of 1947 percent and were also shown to generate small chloride currents due to lack of GABA. The EC50 values for the four glycosides were estimated from 26 to 36 μM. The pentose moiety cleavage led to a substantial decline in anxiety-related behavior (particularly for substance 23-O-acetylshengmanol-3-O-D-xylopyranosides. This compound was used in several in-vivo studies for the examination of its anxiolytic and sedative properties. It increased the number of open entries at 0.6 mg/kg in the EPM test whereas reduced stress-induced hyperthermia at doses of 0.2, 0.6, 2 and 6 mg/kg. In the open field test, this compound reduced the distance traveled at doses of 6, 20 and 60 mg/kg and also increased the time spent in the centre at a dose of 60 mg/kg, while the number of entries into the centre was reduced [60].

The discussion of the structure–activity of triterpenes is not influenced by the lack of comparable structures (scaffolds) compared to the last two subsections, but by the variety of test systems used for their analysis. However, it is possible to compare at least some of the known triterpenes from ginseng and black cohosh. Electrophysiological data showed lower EC50 levels for the three ginseng triterpenes ginsenoside C. Unfortunately, the maximum chloride current stimulation

#### *Natural Products Altering GABAergic Transmission DOI: http://dx.doi.org/10.5772/intechopen.99500*

values were only observed for the two aglycones and were recorded to be 54.1 and 23.3 percent, respectively (at a concentration of 100 M). It can be concluded that the receptor modulation of the glycoside would be of significant concern after examining substance 23-O-acetylshengmanol-3-O-D-xylopyranosides, where the xylose moiety cleavage changed the potentiation of GABA-induced chloride currents from 1692 percent to 64 percent (100 M) and thus into the range of ginseng aglycon. Both compounds 23-O-acetylshengmanol-3-O-D-xylopyranosides and ginsenoside C disclose a four-ring structure with a side chain linked to ring D when contrasting their scaffolds. The prenylate and oxyprenylate side chains have enhanced activity, which is reminiscent of the structure-action-relationship of coumarins. The ginsenoside side-chain will stand for the prenyl moiety in the case of the triterpenes under consideration and that of substance23-O-acetylshengmanol-3-O-D-xylopyranosides for the more active epoxylated form. However, this molecule has additional characteristics that may contribute to its pronounced effect, such as keto-function at position 16 or acetyloxy-group at C-23, which both differentiate the compound from the other slightly less active cycloartanoids [59].

The neurosteroid binding site would be most obvious and consistent with the fact that neurosteroids are the most effective natural GABAA receptor modulators and, in the absence of GABA, are also capable of evoking chloride currents [78]. However, the hydroxy group at position 3 and the keto group at position 17 or 20 are considered to be important for neurosteroid binding activity. As far as the structure of compound is concerned, the keto group may well lead to the binding of the receptor in position 16 instead of position 17, but the fact that the role of the compound almost vanishes with the xylose moiety does not support this theory unless the binding of the neurosteroid site can be improved by the residue of sugar instead of the hydroxy group in position 3. Barbiturates, on the other hand, are also known to activate GABA(A) receptors directly at higher concentrations and the site of barbiturate binding is thought to be similar to that of neurosteroids [79].
