Antioxidant and acetylcholinesterase-inhibitory properties of long-term stored medicinal plants

Background Medicinal plants are possible sources for future novel antioxidant compounds in food and pharmaceutical formulations. Recent attention on medicinal plants emanates from their long historical utilisation in folk medicine as well as their prophylactic properties. However, there is a dearth of scientific data on the efficacy and stability of the bioactive chemical constituents in medicinal plants after prolonged storage. This is a frequent problem in African Traditional Medicine. Methods The phytochemical, antioxidant and acetylcholinesterase-inhibitory properties of 21 medicinal plants were evaluated after long-term storage of 12 or 16 years using standard in vitro methods in comparison to freshly harvested materials. Results The total phenolic content of Artemisia afra, Clausena anisata, Cussonia spicata, Leonotis intermedia and Spirostachys africana were significantly higher in stored compared to fresh materials. The flavonoid content were also significantly higher in stored A. afra, C. anisata, C. spicata, L. intermedia, Olea europea and Tetradenia riparia materials. With the exception of Ekebergia capensis and L. intermedia, there were no significant differences between the antioxidant activities of stored and fresh plant materials as measured in the β-carotene-linoleic acid model system. Similarly, the EC50 values based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay were generally lower for stored than fresh material. Percentage inhibition of acetylcholinesterase was generally similar for both stored and fresh plant material. Stored plant material of Tetradenia riparia and Trichilia dregeana exhibited significantly higher AChE inhibition than the fresh material. Conclusions The current study presents evidence that medicinal plants can retain their biological activity after prolonged storage under dark conditions at room temperature. The high antioxidant activities of stable bioactive compounds in these medicinal plants offer interesting prospects for the identification of novel principles for application in food and pharmaceutical formulations.


Background
The detrimental effects of oxidative stress to human tissues and cells caused by reactive oxygen species (ROS) arising from aging and disease pathogenesis is well documented. Though the human body has inherent antioxidative mechanisms to counteract the damaging effects of free radicals, there is often a need to use dietary and/or medicinal antioxidant supplements, particularly during instances of disease attack. An imbalance between ROS such as singlet oxygen, superoxide anion radical, hydroxyl radical and hydrogen peroxide, and the natural detoxification capacity of the body in favour of the oxidant molecules causes oxidative stress leading to cellular and DNA damage as well as oxidation of low-density lipoproteins [1,2]. Oxidative stress disorders caused by the actions of ROS are associated with many acute and chronic diseases such as inflammation and neurodegenerative conditions including Alzheimer's disease (AD) [3]. Alzheimer's disease, an age-related neurological disorder, is characterised by progressive loss of cognitive ability primarily memory loss, leading to dementia. The main strategy in the clinical treatment of AD involves the maintenance of adequate levels of acetylcholine (ACh) at neurotransmission sites [4]. Thus, the inhibition of acetylcholinesterase (AChE) prevents the hydrolysis of ACh thereby maintaining normal memory function. The consumption of antioxidants is highly correlated with lower incidences of AD [5,6]. As a result, the use of natural compounds with high levels of antioxidants has been proposed as an effective therapeutic approach for AD [5].
Against a background of growing concerns about the toxicity and side effects of many synthetic therapeutic agents, there has been a renewed interest globally, in the search for antioxidants and AChE inhibitory compounds from natural sources, particularly medicinal plants [1,2,[7][8][9][10][11][12][13][14]. Medicinal plants have long been used to treat cognitive memory dysfunction symptoms [4,5,[15][16][17][18][19]. The growing relevance of medicinal plants as possible sources for the discovery of novel antioxidant molecules is often based on their long historical utilisation in folk medicine, especially in developing countries. In addition, the recognised health benefits of medicinal plants emanate from their prophylactic properties [6]. Most notably, traditional practices in the Ayurvedic, Chinese and African medicinal systems are strongly based on prevention and the promotion of good health; hence plant extracts and herbal preparations are regularly consumed as rejuvenators, tonics and/ or nutritional supplements [8]. Traditional medicine practitioners and gatherers often store plants before they are eventually consumed. However, there is a dearth of scientific data on the stability and efficacy of the bioactive compounds in medicinal plants after prolonged storage. In the present study, 21 commonly used South African medicinal plants (Table 1) were investigated for their phytochemical, antioxidant and AChE-inhibitory properties after 12 or 16 years storage in comparison to freshly harvested material. These plants are used in traditional medicine to prevent and/or treat pain-related ailments and infections [20][21][22][23]. Fresh materials were harvested from the same locations and season as the stored materials [21,23] to minimise any differences due to geographical and seasonal effects [24].

Chemicals and reagents
Acetylcholine iodide, AChE from electric eel (type VI-S lyophilized powder), β-carotene, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), galanthamine, gallic acid, catechin and linoleic acid were obtained from Sigma-Aldrich (Steinheim, Germany); butylated hydroxytoluene (BHT) from BDH Chemicals Ltd. (Poole, England); and harpagoside from Extrasynthèse (France). All chemicals and reagents used were of analytical grade. Table 1 shows the scientific names, and voucher specimen numbers of the evaluated plant materials. Following oven-drying at 50°C, plant materials were stored at room temperature (25°C) in brown paper bags in the dark for 12 or 16 years. Fresh plant materials collected from the same locations and season as the stored ones were similarly oven-dried at 50°C. The plants were identified by Dr C. Potgieter and voucher specimens deposited in The Bews Herbarium, University of KwaZulu-Natal, Pietermaritzburg, South Africa.

Plant material and preparation of extracts
Dried plant materials were ground to fine powders and extracted with 50% methanol at 20 ml/g in a sonication bath containing ice-cold water for 1 h for antioxidant and AChE assays. Extracts were then filtered through Whatman No. 1 filter paper, concentrated in vacuo at 40°C and completely air-dried at room temperature in glass vials.
The extraction method described by Makkar [25] was used for phytochemical analysis. Dried plant materials, ground to fine powders (0.2 g), were extracted with 50% aqueous methanol (10 ml) in a sonication bath containing ice-cold water for 20 min. The extracts were then centrifuged at approximately 3000 U/min for 5 min using a Hettich Universal 1200 01 Centrifuge. The supernatants were collected and kept on ice for phytochemical analysis.

Phytochemical analysis
Total iridoid content of the plant material was quantified using the method described by Levieille and Wilson [26]. The calibration curve was plotted using harpagoside as the standard. Total iridoid content for each plant material was expressed in μg harpagoside equivalents (HE) per g dry weight (DW).
For the determination of total phenolic content, the Folin & Ciocalteu [27] method was used with slight modifications [28]. Gallic acid was used as the standard for plotting the calibration curve. Total phenolic content was expressed in mg gallic acid equivalents (GAE) per g DW.
The flavonoid content of the plant materials were quantified using the aluminium chloride colorimetric method [29]. Catechin was used as a standard for the calibration curve. Flavonoid content was expressed in mg catechin equivalents (CE) per g DW.
The butanol-HCl method [25] was used to quantify condensed tannin (proanthocyanidin) content of the plant materials. Condensed tannins (% in dry matter) were expressed as leucocyanidin equivalents were calculated using the formula: Condensed tannins % dry matter ð Þ ¼ A 550nm Â 78:26 Â Dilution factor % dry matter Â 100 where A 550nm is the absorbance of the sample at 550 nm. The formula assumes that the effective E 1% 550 of leucocyanidin is 460 [30]. Free gallic acid and gallotannin contents were evaluated using the rhodanine assay [25,31]. The calibration curves were plotted using gallic acid as a standard. Free gallic acid and gallotannin contents were expressed in μg GAE per g DW.

Antioxidant activity
DPPH free radical scavenging activity The DPPH assay [32] was used to evaluate the free radical scavenging activity of the plant extracts. Methanol was used as a negative control while ascorbic acid and BHT were used as positive controls. Any absorbance due to extract colour was removed by including a background solution with methanol in place of DPPH solution for each extract. Each sample was evaluated in triplicate. The radical scavenging activity (RSA) was calculated using the equation: where А extract , A background and A control are the absorbance readings of the extract, background solution and negative control, respectively at 517 nm. The EC 50 , which is the extract concentration required to scavenge 50% of DPPH free radical, was determined for each extract. Antioxidant activity index (AAI) for each extract was calculated using the equation [33]: β-Carotene-linoleic acid model system The assay was done following the method described by Moyo et al. [34]. Methanol and BHT were used as negative and positive controls, respectively. Each sample was prepared in triplicate. The plant extracts and BHT were evaluated at a final assay concentration of 200 μg/ml. Antioxidant activity (%), measured at t = 120 min, was calculated using the following equations: where A t = 0 is the initial absorbance at t = 0 min, A t = t is the absorbance at time t = 120 min, t = 120 min and R is the rate of β-carotene bleaching.

Acetylcholinesterase inhibitory activity
The AChE assay was performed using the colorimetric method [35]. Each extract was evaluated in triplicate at a final assay concentration of 1.0 mg/ml. Galanthamine at a final assay concentration of 20 μM was used as a positive control. The rate of reaction was calculated for each of the plant extracts, the blank (methanol) and positive control (galanthamine). The percentage inhibition by each plant extract was calculated using the formula: AChE inhibition % ð Þ ¼ 1 À Sample reaction rate Blank reaction rate Â 100

Data analysis
The levels of significant difference between the mean values of stored and fresh plant materials were determined using the t-test (SigmaPlot version 8.0). Regression analysis and the determination of EC 50 values were done using GraphPad Prism software (version 4.03).

Phytochemical analysis
The effects of long-term storage on the total iridoid, phenolic and flavonoid content of the plant materials evaluated are presented in Table 1. Of the 21 fresh and stored plant materials evaluated, the levels of total iridoid present in nine plants were significantly higher in fresh compared to the stored plant materials. The total iridoid contents of stored materials in Acokanthera oppositifolia, Solanum mauritanum and Tetradenia riparia were significantly higher than those of fresh ones. There was no significant difference between the iridoid content of fresh and stored plant materials in approximately 50% of the evaluated plants.
The total phenolic contents of Artemisia afra, Clausena anisata, Cussonia spicata, Leonotis intermedia and Spirostachys africana stored materials were significantly higher than in freshly collected material. With the exceptions of A. afra, D. rotundifolia, T. riparia and T. dregeana (where there was no significant difference between the stored and fresh materials), the phenolic contents of the remaining 15 fresh plant materials were significantly higher than in the stored material. Similarly, a comparison of fresh material and herbarium specimens of three Quillaja species revealed non-significant differences in their phenolic constituents [36]. Remarkably, one of the tested herbarium specimens in the Bate-Smith [36] study was 100 years old.
The flavonoid content was significantly higher in stored A. afra, C. anisata, C. spicata, L. intermedia, T. riparia and Olea europea materials when compared to their corresponding fresh materials. It is noteworthy that the stored materials of the former four species had higher total phenolic contents than their fresh materials perhaps due to their higher flavonoid content compared to the fresh materials. Higher flavonoid contents were observed in 12 fresh plant materials when compared to their respective stored materials. Previous studies  comparing the phenolic constituents of some Dillenia species showed differences in the flavonoid profiles of fresh and herbarium materials as some flavonoids were not detected in the latter [37]. The results suggested that some flavonoids are easily oxidised during the drying process [37]. Table 2 presents the condensed tannin, free gallic acid and gallotannin contents of both the stored and fresh materials of plant species evaluated in this study. No condensed tannins were detected in both fresh and stored materials of A. oppositifolia, Pittosporum viridiflorum and Merwilla plumbea. With the exceptions of Buddleja salviifolia (leaves), Plumbago auriculata and Ziziphus mucronata, the condensed tannin content in the stored plant materials was either significantly higher or not different when compared to the fresh materials. Unlike the stored materials, no condensed tannins were detected in fresh material of A. afra, C. spicata, L. intermedia, Leonotis leonurus and O. europea. Among the 21 species evaluated, free gallic acid was detected in 15 fresh and/or stored plant materials. In most cases, there was no significant difference in the free gallic acid contents of the fresh materials when compared to the stored ones. With the exceptions of A. oppositifolia, A. afra and Ekebergia capensis, the gallotannin content of the stored plant materials was either higher or not significantly different when compared to the fresh ones. It has been shown that phytochemical constituents of medicinal plants, such as alkaloids, flavonoids, volatile oils and amino acids are sufficiently stable to even be detected in herbarium specimens [38]. However, based on the results of the present study, the degree of stability of phenolic compounds seems to be species dependent.

Antioxidant properties
The effect of long-term storage on the radical scavenging activity of 21 plant materials is presented in Table 3. The lower the EC 50 value, the higher the antioxidant activity index and the free radical scavenging activity. At 100 μg/ml concentration, the radical scavenging activity of all stored plant materials (with the exception of Protorhus longifolia) was either significantly higher or not different when compared to the freshly harvested materials. A comparison based on the EC 50 values and antioxidant activity indices revealed a significantly higher radical scavenging activity in 58% of the stored plant materials.
With the exception of A. oppositifolia and B. salviifolia (leaves), the radical scavenging activity of the remaining stored plant materials based on their EC 50 values was not significantly different when compared to the fresh materials. The DPPH radical acts as both the probe and oxidant by accepting electrons from antioxidant compounds in the extract. There is a direct correlation between degree of hydroxylation of the bioactive compounds and DPPH radical scavenging activity [11]. Potent DPPH radical scavenging activities of medicinal plants have also been reported in other studies [11,13,14]. However, the significance of the present study lies in the observed high DPPH radical scavenging activity of aqueous methanol extracts obtained from medicinal plant material after prolonged storage. Table 4 presents the effect of long-term storage on the antioxidant activity of medicinal plant materials evaluated based on β-carotene bleaching model. The β-carotene bleaching assay simulates the oxidation of membrane lipid components and measures antioxidant activity towards linoleic acid [16]. The antioxidant activity of E. capensis stored plant material was significantly higher (almost two-fold) compared to the fresh material.
On the other hand, the antioxidant activity of L. intermedia fresh plant material was significantly higher than that of the stored materials. With the exception of E. capensis and L. intermedia, there were no significant differences between the antioxidant activities recorded in both the stored and fresh plant materials. The retention of antioxidant activity in stored plant material suggests the stability of bioactive chemicals during prolonged storage. The detected bioactivity in the stored plant material provides interesting prospects in the future development of stable food additive compounds. In previous studies, high antioxidant activity from polar extracts of some plants has been attributed to hydrogendonating phenolic compounds and flavonoids [2,16]. However, the identification of specific phenolic compounds responsible for the high antioxidant activity of long-term stored plant materials remains a challenge for future research. Table 4 presents the effect of long-term storage on AChE inhibitory properties of the evaluated plant materials. Stored plant materials of T. riparia and T. dregeana showed a significantly higher AChE inhibition than the fresh ones. There was no significant difference between the percentage AChE inhibition by the stored and fresh materials of the remaining plant species. In general, the evaluated plant species exhibited high AChE inhibitory activity. Interestingly, medicinal plant materials retained AChE inhibitory activity even after prolonged storage (12 or 16 years). The results of the present study confirm the therapeutic value of stored medicinal plants in the pharmacotherapy of AD disease. The AChE inhibitory properties of plant-derived extracts obtained from freshly harvested material have been previously reported [16,32]. Recent studies have demonstrated a direct association between AD and antioxidant activity [16]. However, this is the first report on the antioxidant and AChE inhibitory properties of long-term stored medicinal plants. The present findings are important for traditional systems which are characterised by an holistic approach to health provision, based on the prophylactic properties of medicinal plants [6].

Conclusions
The current study presents evidence that dried medicinal plants stored under dark conditions at room temperature remain biologically active after long-term storage. Extracts of the stored plant material still exhibited potent antioxidant and AChE-inhibitory properties. These findings are significant as some medicinal plants may be utilised long after their time of harvesting. In addition, the prevention strategies practised in the Ayurvedic, Chinese and African medicinal systems often involve regular intake of medicinal plant extracts and/or herbal preparations, which are responsible for counteracting the oxidative stress effects caused by ROS. The high antioxidant activity and stability of the bioactive compounds in these medicinal plants offer interesting prospects for the identification of novel principles for application in food and pharmaceutical formulations. However, in vitro and in vivo safety evaluation of the stored medicinal plants is required.

Competing interests
The authors declare that they have no competing interests.