Abstract
Context: Stachyss species have been used as a medicine for centuries throughout the world. Stachys lavandulifolia Vahl. var. lavandulifolia (Lamiaceae) is of interest to researchers because the constituents such as betulinic acid, oleanolic acid, rosmarinic acid, and ursolic acid are similar to other Stachys species commonly used as an alterative in medicinal preparations.
Objective: The present study investigated the effects of water extract (SLEw) and ethanol extract (SLEe) obtained from S. lavandulifolia (SLE) on the longevity of Drosophila melanogaster Meigen.
Materials and methods: The effects of different concentrations of SLE (Control+DMSO; 4.0; 12.0 and 20.0 µL/100 mL medium) were administered separately to female and male populations of D. melanogaster for control and SLE groups.
Results: In all application groups, each population’s longevity increased, depending on the concentration of SLE. The mean life-span of the extract groups which are applied with SLE(w) was determined to be shorter than the extract groups which are applied with SLE(e). For example, the maximum mean life-span applied with SLE(w) increased from 31.86 ± 0.92 days to 43.21 ± 1.33 days and the maximum mean life-span applied with SLE(e) increased from 31.86 ± 0.92 days to 49.62 ± 1.62 days in females.
Conclusion: These findings demonstrate that the constituents of S. lavandulifolia have great potential as a source for natural health products for D. melanogaster management.
Keywords::
Introduction
Phytotherapy is now accepted as a part of medicine rather than an alternative medicine. It has been used in the treatment of many human and animal diseases including cancer treatment (CitationPhillipson & Anderson, 1989). Even in Germany, where there is less plant diversity, more than 500 plants are utilized for medical purposes. In Turkey, which has a rich diversity of plants, there are approximately 10,000 species and 30% of these species are not found in other countries (CitationBhattacharjee, 1982).
These plants are utilized as regional remedies, but since little is known about the characteristics of these plants, the usage is limited. Stachys lavandulifolia Vahl. (Labiatae) is widely used by the people of south Anatolia (CitationMabberly, 1997) for the treatment of gastrointestinal diseases, stomach aches, and as an appetizer.
In this study, the effects of water and ethanol extracts of S. lavandulifolia aerial parts on the longevity in male and female population of Drosophila melanogaster are evaluated.
Materials and methods
Experimental animals
The flies used in the experiments were Oregon R wild type (w.t.) strain of Drosophila melanogaster Meigen (Diptera; Drosophilidae). This stock had been maintained for many years in the Laboratory at the Department of Biology of the Atatürk University in Erzurum and was, therefore, highly inbred with little genetic variation.
Laboratory conditions
The flies were kept at a constant temperature of 25° ± 1°C on standard medium composed of maize-flour, agar, sucrose, dried yeast and propionic acid (Standard Drosophila Medium, SDM). The flies were kept in darkness, except during the transfers onto fresh medium (usually twice weekly). The humidity of the experimental chamber was 40–60%. The females used in this experiment were virgins.
Plant material
The flowering aerial parts of Stachys lavandulifolia was collected in Erzurum region of Turkey (Eastern Anatolia) near the Palandöken Mountain at 1980 m height in July 2005 during the time of flowering. The plant was identified and a voucher specimen was deposited in the Ata Herbarium (no. ATA-9808) by Meryem Şengül from the Department of Biology, Faculty of Science, Atatürk University, Erzurum, Turkey. The freshly picked flowers and leaves of the plant were shade dried at room temperature for 3 weeks.
Extract preparation
The aerial parts of the plant sample (50 g) were separately extracted with 150 mL ethanol (96%, analytical grade, Merck, Darmstadt, Germany) at room temperature three times. The organic solvent was evaporated to dryness under vacuum at low temperature using a rotary evaporator. To obtain the water extract, 50 g plant sample was kept in 250 mL boiling water for 10 min and filtered. Then the water solution was lyophilized by using a Labconco 117 freeze-dryer (CitationCakır et al., 2003). The dried extract was later dissolved in dimethylsulfoxide (DMSO) (99.9%, Sigma, St. Louis, MO) followed by culture medium and prepared in different concentrations.
The application of Stachys lavandulifolia extract to adult individuals
In this study, the effects of SLE on longevity were studied separately in female and male populations. The experiments were repeated three times. To obtain the same age flies, adult individuals mated in the culture vials including only SDM and prestocks were prepared. On average, 100 individuals were collected from among the same aged female and male flies which were not mated and obtained from pupa. Then, the gathered individuals were put into the empty culture vials and they were starved for 2 h before the SLE application. For the application, two layers of blotting papers were placed into each culture vial, SLE(w) and SLE(e) in different concentrations was absorbed into these papers. Afterwards, the gathered flies put into the application vials were left for 2 h. Following the application, 100 individuals put into one vial for application (separately applied for female and male flies) were placed into the culture vials containing only SDM as 25 × 25. The experiments for both control and application groups were started synchronically. All the vials were kept in appropriate thermal cabins. During the experiments, food was replaced with fresh food twice a week. The number of individuals was counted both at the beginning and at the end of each application day, and the dead individuals were registered and then removed from the culture vials. The application was carried out until the last individual died.
Statistical analyses
The obtained data were analyzed with SPSS version 13.0 (Statistical Package for the Social Sciences Software, SPSS, Chicago, IL). The mean longevity values of the control and application groups were subjected to Duncan’s one-way range test (p<0.001).
Results
In this study, it was determined that the water extract (SLEw) and the ethanol extract (SLEe) obtained from Stachys lavandulifolia (SLE) increased the maximum life- span of male and female population according to control group which belongs to D. melanogaster. It was observed that the maximum female lifespan of the control group was 50 days; the maximum lifespan of males belonging to the control group was 47 days and the maximum male and female lifespan of the Control+DMSO group were 47 and 43 days, respectively. The difference between control and Control+DMSO groups is not statistically significant (p>0.001) ().
Table 1. The longevity of male and female populations of D. melanogaster and the probability levels between groups.
According to results obtained from application groups, in the female population of D. melanogaster applied with SLE(w), the maximum lifespan was 50 days for the lowest application group (4.0 µL) and the maximum life-span 57 days for the highest application group (20.0 µL). It was also found that the maximum male life-span in 4.0 and 20.0 µL application groups were 50 and 54 days, respectively ().
Figure 1. The survivorship lines of female and male individuals of D. melanogaster living medium applied with different concentrations of SLE(w) during adult stages.
In adult populations of D. melanogaster applied with SLE(e), the female life-span within the lowest (4.0 µL) and highest (20.0 µL) application groups is 57–68 days, whereas the male life-span is 50–64 days ().
Figure 2. The survivorship lines of female and male individuals of D. melanogaster living medium applied with different concentrations of SLE(e) during adult stages.
On the other hand, the present results indicated that SLE(e) was more potent than SLE(w) (). When the application groups of the females were compared with control group separately, the maximum mean life-span applied with SLE(w) increased from 31.86 ± 0.92 days to 43.21 ± 1.33 days and the maximum mean life-span applied with SLE(e) increased from 31.86 ± 0.92 days to 49.62 ± 1.62 days. As in the females, the maximum mean life-span applied with SLE(w) increased from 30.93 ± 0.92 days to 40.84 ± 1.35 days, and the maximum mean life-span applied with SLE(e) increased from 30.93 ± 0.92 days to 46.0 ± 1.57 days in the males. The difference observed in terms of mean life-span was statistically significant (p<0.001) in both sexes.
Discussion
In our study, external (environmental) or internal factors that may affect the longevity of the groups were reduced to minimum levels in the application environment. One of these factors is photoperiod and it was determined to be effective during the period of laying egg and coming out of pupa (CitationQiu & Hardin, 1996), on metabolic velocity (CitationLanciani et al., 1991) and the longevity (CitationSheeba et al., 2000) of D. melanogaster. Individuals of control and experimental groups were removed from the incubator only during nutrient exchange and thus the impact of the light on the longevity was corrected. In our study standard drosophila medium (SDM) was used instead of different types of food, which might affect longevity (CitationDavid et al., 1975). Maternal age is known to be an important factor on longevity of offspring (CitationSorensen & Loeschcke, 2002). Therefore, in our experiments, the individuals which were handled as a result of the match between virgin males and females of the same age (3 days) were used. CitationBradley TJ, Simmons FM (1997), determined by ten different populations of Drosophila, ascertained that longevity was increased when metabolic wastes did not exist in the medium. So, the negative effect of waste on the longevity was corrected via frequent renewal of the medium.
The above-mentioned internal and external factors that may affect longevity were fixed during our experiment. The only variable parameter in our experiment was S. lavandulifolia extract, which was practiced at different concentrations.
Plants belonging to the genus Stachys have long been applied to treat various diseases such as sclerosis of the spleen, genital tumors, inflammatory tumors, and cancerous ulcers (CitationSkaltsa et al., 1999). CitationSaeedi et al. (2008) observed that methanol extracts of various Stachys species such as S. byzantina K. Koch, S. inflata Benth., S. lavandulifolia and S. laxa Boiss. & Buhse had antimicrobial effects especially on Gram (+) microorganisms. In addition, CitationHajhasemi et al. (2006) determined analgesic and anti-inflammatory effects of Stachys lavandulifolia extracts on mice. Many studies have confirmed that extracts or components of Stachys species display significant antitoxic (CitationZinchenko et al., 1981), antinephritic (CitationHayashi et al., 1994; CitationSavchenko & Khvorostinka, 1978), anti-anoxia (CitationYamahara et al., 1990), hypotensive (CitationTakeda et al., 1997), and anti-anxious (CitationRabbani et al., 2005) effects.
Phytochemical investigations show that Stachys species consist of flavonoids, terpenes, phenyl ethanoid glycosides, and saponins (CitationKhanavi et al., 2005). CitationJanicsak et al. (2006) found higher amounts of oleonolic and ursolic acids in many Stachys and Salvia species. Both oleonolic acid and ursolic acid, which are rather similar because of the closeness of their chemical structures, have many important pharmacological effects. The literature includes numerous data on anti-inflammatory (CitationSafayhi & Sailer, 1997), antitumor (CitationOvesna et al., 2004), anti-HIV (CitationKashiwada et al., 1998), antimicrobial (CitationMallavadhani et al., 2004.), antifungal (CitationRocha et al., 2004), hypoglycemic (CitationPerez et al., 1998) and antihyperlipidemic (CitationMa, 1982) properties.
α-Pinene, myrcene, β-phellandrene, and β-caryophylene were isolated at different stages of growth as pre-flowering, flowering, and post-flowering of S. lavandulifolia by hydro distillation (CitationMeshkatalsadat et al., 2007). Seventy-nine compounds were identified, representing 98.2% of the essential oil of S. lavandulifolia, in which the major components were germacrene-D, β-phellandrene, β-pinene, myrcene, α-pinene, and Z-β-ocimene (CitationJavidnia et al., 2004). Forty-four components were identified representing 89.6% of the oil of S. lavandulifolia, which was particularly rich in monoterpenes and sesquiterpenes. Among the monoterpene fractions, oxygenated compounds were only in small percentages as compared with the hydrocarbons. α-Pinene, β-pinene, spathulenol, and germacrene-D were found to be the major constituents (CitationFeizbaksh et al., 2003). Also, CitationMortaza-Semnani et al. (2006) identified major components such as 4-hydroxi-4-methyl-2-pentanone, α-pinene, and hexadecanoic acid in the essential oil of Stachys lavandulifolia.
In a study on rats with the α-pinene obtained from the essential oils of Foeniculum vulgare Mill. var. dulce (Apiaceae), it was reported that α-pinene had a protective effect on hepatotoxicity caused by carbontetrachlorore (CitationOzbek et al., 2006).
CitationJafari et al. (2008) found that the extract obtained from Rosa damascena Mill. (Rosaceae) reduced mortality in the adult Drosophila melanogaster. In previous studies, it was determined that the water extract obtained from Usnea longissima Ach. (Ascomycota, Parmeliaceae) had remedial effects on longevity and some development stages of D. melanogaster (CitationUysal et al., 2009a, Citation2009b). CitationUysal et al. (2010) also showed that the extract obtained from Lobaria pulmonaria (L.) Hoffm. (Lobariaceae) increased the longevity of male and female population of D. melanogaster. The extracts of green tea and broccoli increased the activity of antioxidant enzymes thus reducing the high percentage of mortality in D. melanogaster caused by the consummation of fatty nutrients, (CitationMan Li et al., 2008).
The balance between oxidants and antioxidants changes depending on age. Metabolism functions as oxidant by-products of normal energy metabolism extensively damage DNA, proteins, and other molecules in the cell, and this damage accumulates with age. Therefore, antioxidant activity is a basic requirement for life (CitationCook & Samman, 1996). Antioxidant defense systems including antioxidant enzymes, food, and drugs are important for the prevention of many diseases (CitationYen & Hsieh, 1998). By aging, in vivo activities of the antioxidant enzymes (catalase, glutathione peroxidase, etc.) and the levels of reduced glutathione were observed in D. melanogaster. For the organism, the consumption of fruit and vegetables, which are the main source of antioxidants in the diet, may create a protective effect with endogenous enzymatic antioxidant defense systems (CitationAmes et al., 1993). CitationMantle et al. (2000) determined that with the help of flavonoids and saponins, the extracts of the some Stachys species have shown antioxidant activity. The antioxidant activities of these compounds can exert their antioxidant activity through various mechanisms, for example, by scavenging radicals, which initiate lipid peroxidation and lipid peroxide radicals, binding metal ions, and inhibiting enzymatic systems responsible for free radical generation (CitationLebeau et al., 2000).
In the present study we can say that the reason the Stachys lavandulifolia extract groups survived longer than the control group is due to the antioxidant activity of Stachys lavandulifolia mentioned above. Most likely, S. lavandulifolia antioxidant properties cause healing effects on organisms and delay aging. This effect may act by reducing the formation of free oxygen radicals which increase with ageing and preventing oxidative damage.
Conclusions
Longevity and aging are two of the most important issues studied, and are related to all living organisms. Although there is much published regarding the chemical content of Stachys lavandulifolia, there are not many studies on the effects of the chemical content on longevity and aging of living organisms. With this study, the findings obtained may create a basis to analyze the subject further.
Declaration of interest
The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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