Lantana camara ethanolic leaves extracts exhibit anti-aging properties in Drosophila melanogaster: survival-rate and life span studies

Abstract

In this article, we aimed to investigate the anti-aging activity of Lantana camara ethanolic leaves-extract in Drosophila melanogaster: survival and longevity (life span). L. camara leaves were collected and washed thoroughly of sand particles, air-dried, ground, and extracted by the maceration method using ethanol as a solvent. Phytochemical screening was carried out. 168-hour LC₅₀ was determined by exposing fruit flies to concentrations ranging from 5 to 5000 mg/10 g diet for 7-days. Twenty-eight-day survival and longevity studies were carried out by administering L. camara ethanolic leaves extract at 5, 10, and 20 mg/10 g diet to 1–3 days old fruit flies. Each concentration was replicated four times with 50 fruit flies each. The emergence rate of young fruit flies from eggs laid by fruit flies administered L. camara leaves-extracts were also carried out. The total yield of the extraction was determined to be 18%. Phytochemical analysis revealed the presence of alkaloids, Flavonoids, Phenol, steroids, cardiac glycosides, and carbohydrates. 168-hour LC₅₀ of L. camara was also determined to be 1135 mg/10 g diet. L. camara significantly prolonged (P < 0.05) survival rate and extended (P < 0.05) D. melanogaster life span compared with control. L. camara significantly increased (P < 0.05) emergence rate of young fruit flies from eggs laid by fruit flies administered L. camara ethanolic leaves extracts. From the experimental results, it can be concluded that the ethanol extract of L. camara leaves extended the life span of D. melanogaster at these concentrations. Due to similarities of conserved genes between humans and fruit flies, the use of L. camara ethanolic leaves extract at these concentrations is safe and may be recommended as herbal medicine in humans.

Introduction

Aging is a natural process in all living things but its rate is affected by hazardous environmental factors, uptake, and assimilation of dangerous substances and disease [1]. The skin is the tissue most affected by aging in humans; wrinkling of the skin is visible in natural aging [1]. Free radicals are one of the factors that adversely affect aging, and these free radicals are naturally occurring substances in a normal physiological process [2].

Drosophila melanogaster is an appreciable model with a large throughput with some conserved genes that code for human diseases. The fruit fly has become an indispensable model for basic research due to its rapid generation time, up to date genetic tools, and its affordable cost and maintenance in the laboratory [3]. D. melanogaster has been useful as a model in the study of aging [1, 2]. Life span study is the vital measure of the aging rate in drosophila due to rapid generation time and post-mitotic cells that have made this species a good model for aging research [4].

Lantana camara also referred to as “Spanish flag or West Indian Lantana”; and sometimes known as “Red Sage.” L. camara is an “ornamental weed” with aromatic leaves, prange, blue, red, yellow, and bright red flowers, and dark blue and black fruits (drupes) [5]. L. camara especially the leaves has been used as an anti-tumor, antibacterial, anti-hypertensive agent, tonic, and expectorant whereas the roots are known for the treatment of rheumatism, skin rashes, and malaria [5–7]. The infusion method of extraction of the leaves and other parts are known for their anti-inflammatory properties and added to the bath as an anti-rheumatic agent [6]. The decoction prepared from L. camara leaves is mostly used in herbal medicine for wound healing, fever treatment, cough treatment, influenza treatment, stomach ache, malaria, etc. [8]. It has also been recorded that can be used for the treatment of cancers, chickenpox, measles rheumatism, and ulcer [9] L. camara also displays the following activities such as antioxidant, anti-fungal, anti-diabetic, analgesic, and anti-inflammatory [8].

There is little or no information on the anti-aging activities of L. camara. This work aims to generate scientific data on the anti-aging effects of this plant in the D. melanogaster model. We investigated the effect of ethanol extract of L. camara leaves on fruit flies survival rate and life span.

Materials and Methods

Plant Collection and Extraction

L. camara leaves were collected from the senior staff quarters of the University of Jos in August 2019, rinsed to remove the sand, and other particles under running tap water. The leaves were air-dried at room temperature for 7 days, and then reduced to powder using mortar and pestle. About 110 g of powdered leaves of L. camara was extracted in ethanol for 72 h by maceration method and further concentrated to dryness using a water bath (50°C) as described by Alexander et al. [10]. The percentage of total yield was calculated using the formula below [11].

where W₀ = the weight of the initial dried sample, W₁ = the weight of the container alone, and W₂ = the weight of the extract and the container.

Fly Strain

D. melanogaster Harwich strains from the Africa Centre of Excellence were bred and maintained on a simple diet prepared using 100 g corn flour, 20 g baker’s yeast, 12 g Agar-agar, 1 g of methylparaben in 5 ml ethanol, and 1700 ml water. Fly laboratory temperature was kept constant at 25°C.

168-Hours Acute Toxicity of L. camara Ethanolic Leaves Extract

Assay of acute toxicity of L. camara was performed following the method described by Alexander et al. [10]. From 1 to 3 days old fruit flies were anesthetized, counted, and exposed to 5, 10, 20, 50, 100, 250, 500, and 5000 mg/10 g diet in 2 replicates each, with each replicate having 30 adult flies. The fruit flies were allowed to acclimatize for 24 h. The mortality rate was scored every 24 h for 7 consecutive days.

28-day Survival and Life Span Studies

Survival and longevity studies were conducted following the method described by Abolaji et al. [12]. From 1 to 3 days old fruit flies were anesthetized on mild ice chips, counted, and exposed to 5, 10, and 20 mg/10 g diet in quadruplicate with 50 flies each. Fruit flies undergoing treatment were transferred to freshly prepared concentration (diet mixed with extract) at 3-day interval. The mortality rate was scored every 24 h for 28 days as in survival rate and a lifetime as in longevity study.

Emergence Rate

Adult fruit flies were administered 5, 10, and 20 mg/10 g diet of L. camara in triplicate with each vial containing 50 flies for 7 days. Male and female fruit flies, five each were sorted out to mate and lay eggs on a freshly prepared diet with no extract (L. camera leaves) for 24 h. At the elapse of 24 h, adult flies (parents) were transferred off and sacrificed while the Vials (containing newly laid eggs) were kept under constant temperature at 25°C, and observed for the emergence of young flies (offspring) [10].

Phytochemical Screening L. camara Ethanolic Leaf Extracts

Qualitative Phytochemical Screening was carried out using a standard procedure as described by Sofowara [13]. The ethanolic leaf extract of L. camara was screened for the presence of Alkaloids, Phenolic acids, Tannin, Flavonoid, Saponins, Carbohydrates, Cardiac glycosides, Terpenoids, Anthroquinone, and steroids.

Statistical Analysis

Data were expressed and presented as Mean ± SEM (standard error of the mean), one-way analysis of variance was performed on the emergence rate followed by Turkey’s posthoc test. Survival curves and LC₅₀ were analyzed using the Log-rank (Mentel–Cox) and non-linear Regression; Dose–response-X is log (dose), respectively (Graphpad Prism statistical software version 7.04). The results were considered statistically significant at P < 0.05

Results

Total Percentage Yield and Phytochemicals of L. camara Ethanolic Leaf Extract

The initial weight of L. camara leaves materials was 110 g whereas the weight of extract after removing the solvent (ethanol) of extraction was 20 g. The % yield was calculated to be 18% w/w. Phytochemical screening of L. camara ethanolic leaf extract result is presented in Table 1.

Table 1.

Qualitative phytochemical screening of L. camara ethanolic leaf extract

S/n	Phytochemicals	Observation
1	Alkaloids	+
2	Phenolic acid	+
3	Tannin	−
4	Flavonoids	+
5	Saponins	−
6	Carbohydrates	+
7	Terpenoids	−
8	Anthraquinone	−
9	Cardiac glycosides	+
10	Steroids	+

+ Present.

− Absent.

168-hours LC₅₀ of L. camara Ethanolic Leaf Extract in D. melanogaster

The result revealed that L. camara is not lethal up to 1000 mg/10 g but between 100 and 500 mg/g, the mortality became significant (P < 0.05) compared with the negative control. The mortality rate ranged from 7 to 95%. The highest mortality rate was scored in 5000 mg/10 g while the lowest mortality rate was scored in the negative control. 168-hour LC₅₀ of L. camara in D. melanogaster was determined to be 1135 mg/10 g diet as shown in Fig. 1.

Figure 1.

168-hour LC₅₀ of L. camara ethanolic leaf extracts in D. melanogaster.

28-Day Survival Rate of D. melanogaster Administered L. camara Ethanolic Extracts

The survival rate of fruit flies administered L. camara ethanolic leaf extract ranged from 50 to 59%. The highest survival rate was recorded in 10 mg/10 g diet while the lowest survival rate was recorded in the negative control. L. camara increased non significantly (P > 0.05) the survival rate of fruit flies at 5, 10, and 20 mg/10 g compared with negative control as shown in Fig. 2. This result implies that L. camara ethanolic leaf extracts may have some protective activities in D. melanogaster.

Figure 2.

Survival rate of D. melanogaster administered L. camara ethanolic leaf extracts (L. camara prolonged the survival of fruit flies non significantly (P > 0.05) compared with negative control).

Longevity (Life Span) of Fruit Flies Administered L. camara Ethanolic Leaf Extracts

The life span of fruit flies administered L. camara ethanolic leaf extracts significantly outlived (P < 0.05) the negatively controlled flies. L. camara ethanolic leaf extracts-treated flies lived up to 50 days with 33.5, 33.6, and 44.5% percentage increase at 5, 10, and 20 mg/10 g diet respectively whereas the negatively controlled flies lived up to 39 days as shown in Fig. 3. This result implies that L. camara ethanolic leaf extracts may have some anti-aging properties or activities in D. melanogaster.

Figure 3.

Life span of D. melanogaster administered L. camara ethanolic leaf extracts (L. camara significantly extended (P < 0.05) the life span of fruit flies).

Emergence Rate of Young Flies from Eggs of D. melanogaster Administered L. camara Ethanolic Leaf Extracts

The emergence rate of young fruit flies from eggs of adult fruit flies administered L. camara ethanolic leaf extracts ranged from 91 to 100%. The highest emergence rate was recorded in 20 mg/10 g diet whereas the lowest emergence rate was recorded in the negative control. The extracts, at 5, 10, and 20 mg/10 g diet increased emergence rate non-significantly (P > 0.05) compared with negative control as shown in Fig. 4. This result implies that L. camara ethanolic leaf extracts may have fertility-enhanced properties in D. melanogaster.

Figure 4.

Emergence rate of young flies from eggs of D. melanogaster administered L. camara ethanolic leaf extracts.

Discussion

Phenolic compounds are secondary metabolites that are produced through phenylpropanoid metabolization of Penrose phosphate. They are also produced in the shikimic acid of the plants [14]. Phenolic compounds (flavonoids, Phenolic acid, Tannins, Cardiac Glycoside, Steroids, Saponins, and alkaloids) have been reported to exhibit anti-aging, anti-inflammatory, anti-proliferative, antioxidant α-glycosidase, and α-amylase inhibitory activities [14–16]. Cardiac glycoside has been reported to inhibit the four genes that are overexpressed in prostate cancer cells including the inhibitors of apoptosis inhibitor survivin and transcription factors [17]. Phenolic compounds are highly recognized for their curative and preventive potential in some disease conditions; Cancer, Diabetes, cardiovascular diseases, Obesity, Bacteria, and Fungi infections [14–18]. The qualitative phytochemical screen of ethanolic leaf extract of L. camara revealed the presence of Alkaloids, Phenolic acid, flavonoids, cardiac glycoside, Carbohydrates, and Steroids which agree with [19]. These compounds may result in the anti-aging phenomenon in Drosophila either individually or in synergy.

D. melanogaster as a model is mostly appreciable in drug screening and toxicity study due to the conserved genes and biochemical pathways that exist between the species and humans. European Centre for the Validation of Alternative Methods (ECVAM) has approved this species as an alternative model having met with the standard for validation of alternative model known as 3Rs: Reduction, Refinement, and Replacement of laboratory animal [20]. Our 168-hour LC₅₀ revealed that this plant leaves extract is safe for use since its LC₅₀ is 1135 mg/10 g diet in D. melanogaster which agrees with Pour and Sasidharan [21], who reported a high value for LC₅₀ of L. camara in the Artemia salina model and no obvious acute toxicity in adult mice. It has been recorded that plants with a high value of LC₅₀ are less or non-toxic while plants with a low value of LC₅₀ are considered to be very toxic and shortens or reduces life expectancy [22]. The survival rate result further supported the claim that L. camara ethanolic leaves extract is safe to use in folk medicine as it increased though non-significantly (P > 0.05) survival rate of fruit flies administered the extracts. This result agrees with Pour and Sasidharan [21], who reported a very low in vivo toxicity of L. camara leaves extract in the Artemia salina model.

The effect of L. camara on aging was investigated using longevity assay. The group of fruit flies administered L. camara ethanolic leaves extract significantly lived longer (P < 0.05) than the negatively controlled flies. This implies that L. camara ethanolic leaf extract is not only safe for use in folk medicine but prolongs survival and extends life span by functioning in an anti-aging manner.

The emergence rate revealed that L. camara ethanolic leaf extracts had no toxic effect on the emergence rate at the concentration tested in D. melanogaster as it increased the emergence rate of young flies with no significance (P > 0.05) compared with the negative control.

Conclusion

From the experimental results, it showed that L. camara prolonged survival and extended the life span of Fruit flies at these concentrations with no sign of toxicity. It is recommended that the anti-aging activities of each of the phytochemicals of L. camara leaf extract be carried out in D. melanogaster to know the most active component.

Conflict of Interest

The authors declare no conflict of interest.