Phytochemical, physicochemical, microbiological study and anticholinesterase activity of Ginkgo biloba L. and Bacopa monnieri L. used in phytotherapy

Herbal medicines based on Ginkgo biloba L. and Bacopa monnieri L. are used to improve memory and cognitive function. The quality control and anticholinesterase activity of the herbal medicines prepared from G. biloba and B. monnieri were evaluated which are commercialized in handling pharmacies. Samples of herbal medicines based on G. biloba and B. monnieri were obtained from handling pharmacies in Teresina-PI, submitted to labeling analysis and anticholinesterase activity. The phytochemical study was performed by preliminary prospecting, TLC and HPLC. The tests physicochemical and microbiological analyses were made according to the Pharmacopoeia 2010. It was observed that the labeling, foreign material, disintegration, and microbiological parameters were in accordance with ANVISA standards. The colorimetric tests were uniform in the samples of B. monnieri and variable of G. biloba. The average weight evaluation shows that the G1 and B3 samples have capsular content above that described in the labeling and presented pH values different. Among the thermogravimetric profiles, the mass losses of samples G1 and B3 showed values with high ash content, suggesting adulterations. Phytochemical prospecting showed flavonoids as common secondary metabolites in both species, corroborating with TLC and HPLC analysis, which identified the compounds chlorogenic acid, rutin, myricetin and quercetin. The samples G4, B2 and B5 present metabolites capable of inhibiting the enzyme acetylcholinesterase with IC50 of 0.8540 mg/mL, 0.9650 mg/mL and 1.8221×10 mg/mL, respectively. The samples G1 and B3 of G. biloba and B. monnieri, did not obey some parameters of quality control for herbal medicines according to the criteria of the Brazilian Pharmacopeia.


Resumen
Los medicamentos a base de hierbas a base de Ginkgo biloba L. y Bacopa monnieri L. se utilizan para mejorar la memoria y la función cognitiva. Se evaluó el control de calidad y la actividad anticolinesterasa de los medicamentos herbarios preparados a partir de G. biloba y B. monnieri vendidos en farmacias de manipulación. Se obtuvieron muestras de medicamentos herbales a base de G. biloba y B. monnieri de las farmacias distribuidoras de Teresina-PI,

Introduction
Phytotherapy is a powerful natural resource for the prevention and treatment of diseases through medicinal plants, used as complementary therapy (Chakraborty, 2018). The Agência Nacional de Vigilância Sanitária (ANVISA), defines herbal medicine as the medicine produced exclusively with derivatives of plant drugs, without the addition of isolated substances in its composition (Brasil, 2010).
Ginkgo biloba L. (Ginkgoaceae), is among the most consumed medicinal plants in the world, has neuroprotective, vascular and cardiological applications, acting against depression, Alzheimer's disease (AD) and ischemic stroke (Yang et al., 2016;Tian et al., 2017;Dai et al., 2018). It has been used as a herbal product in the treatment of patients with AD, as it is considered a potent inhibitor of acetylcholinesterase (AChE) (Kim et al., 2016). Some studies indicate that secondary metabolites isolated from the leaves of this plant, present several biological activities (Singh et al., 2008). In the group of flavonoids stand out, canferol, quercetin, isorhamnetin, myricetin, 3'-methyl-myricetin, catechins and proanthocyanidins (Ding et al., 2008). It also contains terpenic lactones, particularly ginkgolides A, B, C, J and M and bilobalide (Ding et al., 2008;Song et al., 2010). The standardized extract of G. biloba L. EGb761, according to RDC 89/2004(Brasil, 2004, contains at least 24.0% flavonoids and 6% terpenoids. Bacopa monnieri L. (Scrophulariaceae), also known as Brahmi, is a medicinal plant, with branched leaves and purple flowers, known as "memory booster" (Russo & Borrelli, 2005;Rao et al., 2012) This plant has activity against Parkinson's disease, Alzheimer's, epilepsy, behavioral deficit, stress and has an antioxidant effect (Mathew et al., 2010;Jadiya et al., 2011;Saini et al., 2012). The compounds responsible for pharmacological effects include alkaloids, flavonoids, saponins and sterols (Azad et al., 2012;Le et al., 2013). Bacoid saponins A and B are the components responsible for B. monnieri ability to increase the transmission of nerve impulses. It is also responsible for the competence of neuronal repair, increased kinase activity, neurogenesis and restoration of synaptic activity (Azad et al., 2012;Dutta & Chakraborty, 2020). The use of herbal medicines in the treatment of specific cognitive problems search for the identification of AChE enzyme inhibitors.
In this study, the phytochemical, physical-chemical, microbiological profile and anticholinesterase activity of herbal medicines based on G. biloba and B. monnieri commercialized in handling pharmacies in Brazil in the city of Teresina -PI were evaluated.

Sample Selection
Samples of the herbal medicines G. biloba (120 mg) and B. monnieri (225 mg) were purchased from four master pharmacies located in the city of Teresina-PI. The manipulation pharmacies chosen were those that supplied the two herbal Research, Society andDevelopment, v. 10, n. 3, e39010313480, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.13480 4 medicines. Each sample obtained contained 60 capsules, totaling four samples of G. biloba (G1, G2, G3, G4) and four samples of B. monnieri (B1, B2, B3, B4). The G5 and B5 samples were dry leaves and aerial parts of the respective medicinal plants and purchased at a natural products store in the city of Teresina -PI (Brazil), which served as a standard for comparing results with herbal medicines, as shown in Table 1.

Labeling Analysis
The analysis of the sample labels was performed by comparing the information contained in the sample labels with those recommended by RDC No. 67/2007 (Brasil, 2007).

Physicochemical Analysis
All samples were subjected to the analysis of foreign material, determination of pH, disintegration time, and average weight, according to the general methods of the Brazilian Pharmacopoeia (2012).

Thermal Analysis by Thermogravimetry
The dynamic thermogravimetric (TG) curves of G. biloba and B. monnieri samples were obtained in a thermogravimetric apparatus TGA-50 model from Shimadzu, with a heating rate of 10 ºC/min, in the range temperature of 25 ºC to 700 ºC. The analyzes were carried out under a air atmosphere, with a flow of 50 mL.min -1 . 5 mg samples (± 0.05) were placed in a platinum crucible. The TG curves were analyzed using Shimadzu's TA 50 software, to characterize the stages of mass loss. The moisture content was identified from the first stage of mass loss between 25 and 200 ° C. The ash contents were obtained directly from the percentage of the thermal decomposition product at 700 ºC.

Colorimetric Analysis
The colorimetric analysis was made with a portable digital colorimeter with an 8 mm caliber (model FRU®, WR-10QC) in the CIE 10º standard observer, luminance measurement range (L) from 0 to 100 and using the difference formula of color: ΔE a* b* in the CIELAB color space defined by the International Lighting Commission (ILC) in 1976 (McLaren, 2008).

Phytochemical Prospecting
For the phytochemical characterization of the samples, classical tests were carried out to identify the main groups of active ingredients (Matos, 2009): flavonoids; saponins; tannins; steroids/ triterpenoids and alkaloids. For the analysis by thin layer chromatography (TLC), the ethanolic extracts were obtained using 500 mg of the samples with 10 mL of ethanol and applied on silica gel chromatographic plates. Together with the standards of epicatechin, rutin, quercetin, lupeol and sitosterol, the samples was eluted in the following solvent systems: hexane/ethyl acetate (80:20), chloroform/methanol (90:10) and chloroform/methanol/water (65:30:5). The spots were visualized by spraying with ceric sulfate and placed on a hot plate (Chaves, 1997).

Phytochemical Profile by HPLC Analysis
The Initially, ethanolic extracts were prepared from the samples of G. biloba and B. monnieri using 500 mg of the samples diluted in 10 mL of ethanol. Ethanol extracts obtained from herbal medicines and standard solutions of gallic acid, catechol, catechin, chlorogenic acid, caffeic acid, (-) epicatechin, syringaldehyde, cumaric acid, coumarin, rutin, myricetin, and quercetin were filtered on a Macherey-Nagel chromabond® C18-ec solid-phase extraction cartridge and 0.45 μm filter disc.
The samples were filtered on 25 mm Chromafil® Xtra PTFE-20/25 filter membrane with 0.20 µm pore. Elution process was performed with a 0.1% acetic acid solution in water (Solvent A) and methanol (Solvent B).
The extract and standards were eluted according to solvent gradient B: 0 to 5 min (7-11%); 5 to 10 min (11-16%); The compounds present in the ethanolic extracts were identified by comparing retention time (Rt) with those obtained by injecting standards prepared under the same conditions. For quantification, an analytical curve (10 points) was constructed from a solution containing a mixture of all standards (Mix). For this, solutions with concentrations ranging from 1 to 10 ppm in water: ethanol solution (70:30 v/v) were prepared. The quantification of compounds was executed by correlating the signal area and added standards volume, being the concentration expressed in g.Kg -1 .

Microbiological Analysis
Previously, Sabouraud Agar (fungi), Xylose Lysine Deoxycholate Agar (Salmonella), MacConkey Agar (Escherichia coli), and Salt Mannitol Agar (Staphylococcus aureus), the buffer solution and the dilution of the samples (1:10) were prepared. This was followed by the inoculation on the surface of the media in plates and their incubation for 24 hours at 35 °C (bacteria) and 7 days (fungi). After the indicated period, macroscopic observation and colony counting was performed according to the Brazilian Pharmacopeia (2012).

Enzymatic Assay of Acetylcholinesterase
The enzymatic assays for AChE inhibition were carried out according to the methodology proposed by Ellman et al. (1961), modified by Rhee et al. (2001). The procedures were performed in triplicate. The percentage of inhibition, a concentration that inhibits 50% of the enzyme (IC50), was calculated using the equation: % inhibition = 100 -(A sample/A white) x 100; Where, A is the change in absorbance at the beginning and end of the readings. Data were calculated using the GraphPad Prism Data Editor for Windows, version 6.0 (GraphPad Software Inc., San Diego, CA).

Labeling Analysis
The RDC nº 67/2007 (ANVISA), establishes several operational procedures for the labeling of developed products, such as every preparation must contain relaiable and correct information, that would be provided to the patient (Silva and Silva, 2014). The results for the labeling investigation of G. biloba and B. monnieri phytotherapeutics from the referred study showed that all samples are in compliance with the current legislation, showing no inconsistencies with the information presented and required.

Physico-chemical analysis
The purity and quality of the samples of G. biloba and B. monnieri showed that the characteristics of the samples of G. biloba did not show similarities with the vegetal sample G5, whereas the samples of B. monnieri were more homogeneous in relation to the plant sample B5. The analysis of the foreign material revealed that only the G3 sample had 0.03% impurities, corresponding to structures similar to white crystals, as shown in Table 2.
However, this sample passed the quality test, since the material index strange present did not exceed that allowed by the Brazilian Pharmacopeia (2012), which is a maximum of 2% for plant medicines. By the values of the phytotherapic contents, of G. biloba and B. monnieri samples, described in Table 3, it is observed that the G2 sample had an average phytotherapic content close to 120 mg, while G1 presents twice the concentration expressed on the label.
Through values of herbal contents, the average weight, relative standard deviation, and limit of variance were calculated for G. biloba and B. monnieri samples. Table 3 shows that the G2 sample had an average phytotherapic content  Table 3.   * Average of three repetitions ± standard deviation (SD) and s* = unit of time in seconds. a,b . Source: Authors.

Thermal analysis by thermogravimetry (TG) curves
The thermogravimetric characteristics of samples G2, G3 and G4 showed similar profiles, which suggests that the samples have the same composition ( Figure 2a). However, the G1 sample differs from the others, suggesting the presence of other constituents in addition to the encapsulated composition. This result corroborates the discrepant data of mean weight and pH (Table 3), presented by the G1 sample. The analyzes of samples B1, B2 and B4 suggest uniformity between the profiles, evidenced by the decomposition steps, indicating classes of similar organic compounds for the samples of B. monnieri ( Figure   2b). Only sample B3 had different capsular content and pH than other samples of B. monnieri (Table 3).
The study by Macêdo (2020), states that thermal analysis is an important tool used in the quality control of herbal products, allowing the simultaneous verification of moisture content, ash and access to the entire thermoanalytical profile of the samples. According to the values established by the Brazilian Pharmacopeia (2012), it was observed that the moisture content, obtained by thermogravimetry, for all samples of G. biloba and B. monnieri were below the maximum limit of 14.0%.
On the other hand, regarding the ash contents of the samples of G. biloba and B. monnieri, only samples G2 and G4 were approved, with values below the maximum limit of 2.0%. Samples G1 and B3 had the highest ash content of 50.59% and 13.15%, respectively, suggesting adulteration or production error (Table 4).

Colorimetric Coordinates Analysis
In the colorimetric data presented in this work, it was indicated that the variation of shades of the solid samples of G. biloba and B. monnieri are confirmed by the gradual reduction in the L * coordinate, presenting phytotherapics with a darker or more intense color (Tosun et al., 2008) It was observed that the L * values were not discrepant in the sampling evaluation of herbal medicines based on B. monnieri, showing uniform color and that the samples have the same formulation pattern, as shown in Table 2. The values obtained for the chromatic coordinate or blue-yellow matrix (b *) indicate that the samples of G. biloba and B. monnieri show uniformity and tendency to develop yellow color (Esteves et al., 2008;Pincelli et al., 2012).

Phytochemical study
In the phytochemical analysis of ethanolic extracts, the samples of G. biloba revealed the presence of flavonoids and the samples of B. monnieri showed positive results for flavonoids and triterpenes, as is evidenced by Figures 3 (c) and 4 (c,d). Ibrahim and Nuhu (2016), showed that the main classes of bioactive metabolites identified in G. biloba extracts were the flavonoids and terpenes that are responsible for the antioxidant activity of this plant. The secondary metabolites that confer the pharmacological activities of B. monnieri are saponins, flavonoids, phenols, alkaloids, terpenoids, tannins and steroids (Azad et al., 2012). The chromatographic profile allows, in many cases, confirmation of the identity of herbal medicine and even the detection of forgeries (Sherma & Rabel, 2020).
In Figure 3 (a,b), the TLC analyzes of G. biloba samples showed that the main constituents identified were the flavonoids. Samples G1, G2, G3 and G4 demonstrated yellow spots with Rf close to the standard quercetin flavonoid ( Figure   3a). However, in Figure 3b, it can be observed that samples G2, G3 and G4 displayed yellow spots with Rf close to the standard rutin flavonoid. These results corroborate the findings of the preliminary phytochemical analysis (Figure 3c). Banov (544.30 g/kg), G3 (1023.14 g/kg) and G4 (1003.72 g/kg) and the quercetin (2) compound in sample G1 (608, 00 g/kg) ( Figure   5), corroborating the TLC results of this study (Figure 3a, b).
The comparison of the chromatographic profiles determined by HPLC of the herbal medicines (B1, B2, B3 and B4) and the plant sample (B5) of B. monnieri showed similar phytochemical composition, which is evidenced by the presence of several peaks with times of distinct retentions. In the samples of B. monnieri, traces of phenolic compounds were identified and quantified, such as chlorogenic acid, rutin, myricetin and quercetin ( Figure 6). Sample B1 stood out with the presence of the compounds rutin (1) (3.71 g/kg), quercetin (2) (3.50 g/kg), chlorogenic acid (3) (2.36 g/kg) and myricetin (4) (4.57 g/kg).
The remaining samples had only two or one of these compounds.
The flavonoids rutin and quercetin, identified in the samples of G. biloba and B. monnieri, as well as steroidal saponins may also be the secondary metabolites responsible for the efficacy of these herbal medicines in the treatment of memory loss. Rutin has pharmacological and therapeutic importance because it inhibits the process of free radical formation, in addition to preventing damage to the nervous system by modulating the activity of enzymes such as the enzyme acetylcholinesterase (Youdim et al., 2003;Soobrattee et al., 2005;Park, 2010). Quercetin has broad therapeutic benefits that involve its antioxidant and anti-inflammatory effects (Oliveira & Ropke, 2016) and its neuroprotective activity (Lu et al., 2006). Quercetin is capable of preventing memory and learning loss and reversing cognitive deficits induced by AD Gayoso et al., 2017).

Reddish color indicating Flavonoids
Phytochemical Prospecting C D

Microbiological Analysis
Microbiological tests assess the quality of the finished product in order to rule out possible contamination by bacteria and fungi, which are extremely pathogenic, and may irreversibly worsen the clinical condition of the use of herbal medicine (Andrade et al., 2013;Bonfilio et al., 2013). G. biloba and B. monnieri samples did not show growth of bacteria such as Escherichia coli, Staphylococcus aureus and Salmonella spp., therefore they are in accordance with the parameters described in the Brazilian Pharmacopoeia, which recommends a value of up to 10 4 CFU.mL -1 for approval. In relation to fungal growth, all samples analyzed were within the required quality standards (up to 10 2 CFU/mL).
Therefore, both samples analyzed are in accordance with RDC 67/2007-ANVISA. The acceptable values of microorganisms in the analyzed samples may be associated with the antimicrobial activity described for the vegetable species G. biloba and B. monnieri. According to Ibrahim and Nuhu (2016), the GbE 761 extract has a considerable inhibitory activity against pathogenic bacteria and fungi, which may be due to the presence of varieties of active compounds in the extract such as flavonoids and tannins. The study by Fazlul et al. (2019) revealed that extracts of B. monnieri have demonstrated broadspectrum antimicrobial activity, with flavonoids, tannins and phenolic compounds responsible for this inhibitory action.

Anticholinesterase activity
Several reversible AChE inhibitors are used in clinical trials as drugs to treat AD (Zhanga et al., 2018). Standardized extract of the leaves of G. biloba, labeled GbE 761, has been widely used since its introduction in the market to improve cognitive deficits in a wide range of conditions, from aging to dementia (Müller et al., 2019). Pre-clinical in vitro, in vivo and clinical studies, have confirmed the neuroprotective effects of GbE 761, being considered the most sold herb for various health disorders (Ibrahim & Nuhu, 2016).
Studies suggest that aerial parts of B. monnieri have the potential to improve cognitive function, possibly by reducing AChE activity, increasing antioxidant function, improving cerebral blood flow and neurotransmitter modulation (Kongkeaw et al., 2014). Vinutha et al. (2007), in their study of AChE inhibition with extracts of medicinal plants, proposed the classification of the inhibitory potential of the samples: strong inhibitors (> 50% inhibition), moderate inhibitors (30-50% inhibition) and weak inhibitors (<30% inhibition).
Thus, samples G4, B2 and B5 with percentage values above 50%, were classified as strong AChE inhibitors. In this research, the study of the anticholinesterase inhibitory activity showed that of the five ethanolic extracts of G. biloba tested, only G4 showed an inhibition above 50% (59.92% ± 14.00) with IC50 of 0.8540 mg/mL (Table 5). The promising values of quantitative AChE inhibition were observed in the ethanol extracts of B. monnieri, for samples B2 (56.03% ± 5.96) and B5 (98.61% ± 0.46), with an IC50 of 0.9650 mg/mL and 1.8221x10 -5 mg/mL, respectively (Table 5).
These results are relevant, with the B5 sample being more effective when compared to rivastigmine IC50 = 1.87x10 -3 mg/mL (Cavalcante et al., 2018), which is an AChE inhibitor drug widely used in the treatment of people with AD. AChE was more inhibited in the presence of G. biloba compared to B. monnieri, and none of the extracts of B. monnieri showed more than 50% inhibition, which differs from our results suggesting that the quantitative AChE inhibition may be an improvement in disturbed cholinergic function (Ramasamy et al., 2015). B. monnieri has been reported as a direct inhibitor of AChE activity tested by an in vitro enzymatic assay (Le et al., 2013). In the report by Yamchuen et al. (2017), B. monnieri extract and all tested compounds did not alter the basal cell activity of AChE.  The AChE inhibition test showed that the G3 sample is promising when compared to the G5 vegetable sample of G. biloba. In relation to the samples of B. monnieri, the vegetal sample B5 showed an inhibition potential of 98.61%, reinforcing the importance of this phytotherapy in the improvement of cognitive processes.